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Molecular structure of α, β, [delta] γ-tetraphenylporphinatoindium(III) chloride, and perturbed angular… Lee, Kai Mon 1979

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MOLECULAR STRUCTURE OF a,6,6,y-TETRAPHENYLPORPHINATOINDIUM(III) CHLORIDE, AND PERTURBED ANGULAR CORRELATION STUDY ON RECONSTITUTED MYOGLOBIN by KAI MON,\LEE B. Tech. (Hons.), U n i v e r s i t y o f B r a d f o r d , E n g l a n d , 1977, Grad RIC  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in CHEMISTRY THE FACULTY OF GRADUATE STUDIES (Department  of Chemistry)  We a c c e p t t h i s t h e s i s as conforming to t h e r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA June, 1979 ©  K a i Mon, L e e , 1979  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f an a d v a n c e d d e g r e e a t the L i b r a r y I further for  shall  the.University  make i t  agree that  thesis for  It  financial  Department  gain shall  of,  The U n i v e r s i t y o f B r i t i s h 2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5  Columbia  I agree  r e f e r e n c e and  that  not  for  that  study.  this  thesis  by t h e Head o f my D e p a r t m e n t  i s understood  written permission.  Columbia,  extensive copying of  s c h o l a r l y p u r p o s e s may be g r a n t e d  this  British  freely available for  permission for  by h i s r e p r e s e n t a t i v e s . of  of  the requirements  or  copying or p u b l i c a t i o n  be a l l o w e d w i t h o u t  my  -iiABSTRACT  The f i r s t  c r y s t a l and m o l e c u l a r  s t r u c t u r e of an indium p o r p h y r i n ,  a,$,6,Y-tetraphenylporphinatoindium(III) determined  by X-ray d i f f r a c t i o n .  square-pyramidal  chloride  The indium  (InTPP:Cl) has been  i s five-coordinate i n a  complex w i t h c h l o r i d e as the a x i a l l i g a n d .  The  o  average  o  In-N bond i s 2.156(6)A, w i t h an I n - C l d i s t a n c e o f 2.369(2)A.  The p o r p h i n a t o c o r e i s somewhat expanded w i t h an average  radius of  o  o  2.067(3)A, and t h e macrocycle  i s non-planar  w i t h a net doming  (0.1A)  o  toward the indium.  The indium atom i s l o c a t e d 0.61A above the mean  p l a n e o f the f o u r p y r r o l e n i t r o g e n atoms. Based on the h i g h degree o f analogy between t h e p r i n c i p a l s t r u c t u r a l f e a t u r e s of InTPP:Cl,  F e ( I I I ) p o r p h y r i n s , and F e ( I I )  deoxyhemoglobin (e.g., displacement  o f metal atom above t h e mean  p o r p h y r i n p l a n e , d i s t a n c e from t h e m e t a l t o p y r r o l e n i t r o g e n , p o r p h y r i n c o r e expansion,  doming of p o r p h y r i n s k e l e t a l c o r e atoms),  the present study suggests  t h a t r e c o n s t i t u t i o n o f an indium  porphyrin  i n t o apohemoglobin and apomyoglobin should produce t h e quaternary "tense" conformation.  T h i s p r o v i d e s an unique  o p p o r t u n i t y to study  the p o r p h y r i n - a p o p r o t e i n i n t e r a c t i o n s i n the "T" s t a t e o f hemoglobin and  myoglobin. The p r e s e n t r e c o n s t i t u t i o n of i n d i u m - I l l meso-protoporphyrin  (^^InMPP IX) i n t o myoglobin r e p r e s e n t s t h e f i r s t  l o c a t i o n of a motional  probe a t t h e c e n t r a l metal o f t h e a c t i v e s i t e o f the p r o t e i n . r o t a t i o n a l c o r r e l a t i o n time been determined technique.  IX  The  ( T ) o f t h e r e c o n s t i t u t e d myoglobin has c  by the P e r t u r b e d Angular  C o r r e l a t i o n s (P.A.C.)  S i n c e . ^"^InMPP IX i s s t r u c t u r a l l y s i m i l a r to f e r r o -  p r o t o p o r p h y r i n IX (heme), t h e r e should be v e r y l i t t l e i n t e r n a l  rotation  -iii-  of the  InMPP IX a t t h e a c t i v e s i t e o f t h e r e c o n s t i t u t e d m y o g l o b i n .  Furthermore, the i n d i u m - l a b e l l e d myoglobin i s very l i k e l y t o r e t a i n i t s n a t i v e conformation,  i n contrast t o other l a b e l l i n g  techniques  i n w h i c h t h e r e i s always u n c e r t a i n t y as how much t h e l a b e l the s t r u c t u r e o f t h e p r o t e i n b e i n g additional f l e x i b i l i t y  s t u d i e d . F i n a l l y , s i n c e no  i s introduced  p r o t e i n , the present determination  distorts  at the l a b e l l e d s i t e of the  o f T ( P . A . C . ) f o r m y o g l o b i n i s more c  d i r e c t t h a n f l u o r e s c e n c e d e p o l a r i z a t i o n o r ESR  determinations.  -ivTABLE OF CONTENTS Page ABSTRACT  i  TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES ACKNOWLEDGEMENTS CHAPTER I . GENERAL INTRODUCTION P a r t One. C r y s t a l and M o l e c u l a r S t r u c t u r e o f a, 3 , Y > <5-Tetraphenylporphinatoindium (III) Chloride CHAPTER I I .  i iv  v i i x i x i i 1 6  7  2.1  Metalloporphyrin Stereochemistry  7  2.1a  Iron Porphyrins  18  2.1b  Cobalt Porphyrins  21  2.2  Application of Metalloporphyrin S t e r e o c h e m i s t r y t o Heme S t e r e o c h e m i s t r y i n Hemoproteins (Myoglobin and Hemoglobin)  24  2.3  M y o g l o b i n and Hemoglobin - The Heme Group/Globin S t r u c t u r e  34  2.4  Models f o r M y o g l o b i n and Hemoglobin  41  CHAPTER I I I . EXPERIMENTAL WORK 3.1  P r e p a r a t i o n o f a ,3 ,y,(S-Tetraphenylporphinatoindium(III) Chloride  45  3.2a  P u r i f i c a t i o n o f a,g,Yj<$-Tetraphenylporphinatoindium(III) Chloride  48  3.2b  T h i n - l a y e r Chromatography  48  3.2c  V i s i b l e A b s o r p t i o n Spectroscopy  48  3.2d  N u c l e a r Magnetic Resonance S p e c t r o s c o p y  51  3.3  R e c r y s t a l l i s a t i o n of a,3,y,6-Tetraphenylporphinatoindium(III) Chloride  51  -V-  Page  RESULTS AND DISCUSSION  52  4.1  Metal Displacements Above Mean Pyrrole Nitrogen Plane  52  4.2  Bond Lengths and Angles  61  4.3  Doming of the Porphyrin Skeleton  4.4  Phenyl Rings: Crystal Packing  CHAPTER IV.  Part Two. Perturbed Angular Correlation Study on Myoglobin (Radioactive ^ - I n - l a b e l l e d porphyrin/  62 65 75  Reconstituion/T determination) c  CHAPTER V.  PERTURBED ANGULAR CORRELATION GENERAL INTRODUCTION  CHAPTER VI. THEORY OF PERTURBED ANGULAR CORRELATION OF NUCLEAR RADIATION  76  79  6.1  Introduction  79  6.2  Theoretical Consideration  82  6.2a  Time-Dependent Quadrupole Interactions The Limit of Rapid Motion  90  6.2b  Static Electric Field Quadrupole Interactions - Polycrystalline Samples  95  6.2c  Time-Dependent Quadrupole Interactions The Limit of Slow Motion  99  6.2d  Fields Without Axial Symmetry  106  CHAPTER VII. APPLICATIONS OF GAMMA-GAMMA CORRELATIONS TO BIOLOGICAL MACROMOLECULES  111  CHAPTER VIII.EXPERIMENTAL WORK  118  8.1  Preparation of Meso-Protoporphyrin IX  118  8.2  Preparation of Non-Radioactive Indium-III Meso-protoporphyrin IX  123  8.2a  Thin-layer chromatography  124  -vi-  Page  8.2b  P u r i f i c a t i o n of Indium-III M e s o - p r o t o p o r p h y r i n IX  123  8.2c  V i s i b l e A b s o r p t i o n Spectroscopy  125  8.3  Preparation of Radioactive Indium-Ill M e s o - p r o t o p o r p h y r i n IX  128  8.A  P r e p a r a t i o n o f Apo-rayoglobin S o l u t i o n  130  8.5  R e c o n s t i t u t i o n o f Apo-myoglobin w i t h I n d i u m - I l l M e s o - p r o t o - p o r p h y r i n IX  132  8.6  P e r t u r b e d A n g u l a r C o r r e l a t i o n Measurements  135  8.6a  Detectors  135  8.6b  Electronics  136  8.6c  Method  138  RESULTS AND DISCUSSION  141  9.1  R e s u l t s - T i m e - D i f f e r e n t i a l Perturbed Angular C o r r e l a t i o n Data  141  9.2  Discussion  149  9.2a  A n a l y s i s of t h e T i m e - D i f f e r e n t i a l P e r t u r b e d Angular C o r r e l a t i o n Spectra  149  9.2b  Least-Squares Analyses of the T i m e - D i f f e r e n t i a l P e r t u r b e d A n g u l a r C o r r e l a t i o n Data  151  9.2c  Comments on t h e R o t a t i o n a l C o r r e l a t i o n Times of M y o g l o b i n determined by t h e P e r t u r b e d A n g u l a r C o r r e l a t i o n s Technique  153  CHAPTER IX.  Appendix  A.  C o - o p e r a t i v e E f f e c t o f R e v e r s i b l e Oxygenation i n hemoglobin  164  Appendix  B.  Tetraphenylporphine Diacid  167  Appendix  C.  The E f f e c t o f E x c i t a t i o n o f t h e E l e c t r o n S h e l l on A n g u l a r C o r r e l a t i o n  169  References  172  -viiLIST OF FIGURES  Figure  Title  2.1  The n u c l e u s of p o r p h i n e , i l l u s t r a t i n g the p o s i t i o n s o f p o s s i b l e s u b s t i t u e n t s and the n o t a t i o n used f o r the unique atoms of the core  2.2  The p e r i o d i c t a b l e of m e t a l l o p o r p h y r i n s  2.3  A diagram of the s q u a r e - p y r a m i d a l c o o r d i n a t i o n group f o r f i v e - c o o r d i n a t e m e t a l l o p o r p h y r i n s  2.A  E n e r g y - l e v e l diagram f o r coordination  2.5  C a l c u l a t i o n of the "doming" of the p o r p h i n a t o  2.6  I l l u s t r a t i o n of c a l c u l a t i o n of the t o t a l movement of the p r o x i m a l h i s t i d i n e i n hemoglobin and Co-hemoglobin  2.7  I l l u s t r a t i o n of p o s s i b l e p o i n t s of d i s t o r t i o n of C o ( I I ) p o r p h y r i n p r o s t h e t i c group  2.8  F e r r o - p r o t o p o r p h y r i n IX (heme) and i t s c o o r d i n a t e system  2.9  Attachment of heme i r o n t o N  2.10  A three-dimensional myoglobin  2.11  The  2.12  A schematic  2.13  "Picket-fence" porphyrin  2.1A  "Capped" p o r p h y r i n  2.15  C h e l a t e d hemes h a v i n g v a r y i n g degrees of s t e r i c h i n d r a n c e toward base c h e l a t i o n  3.1  V i s i b l e a b s o r p t i o n spectrum o f InTPP:Cl chloroform  3.2  V i s i b l e a b s o r p t i o n spectrum o f ineso-TPP i n chloroform  A.l  Atomic numbering scheme f o r c r y s t a l l i n e  A.2  S t e r e o s c o p i c v i e w o f the c o n t e n t s of one u n i t c e l l of c r y s t a l l i n e InTPPrCl  square-pyramidal  of h i s t i d i n e  core  F8  s t e r e o - p a i r drawing of  f o l d i n g and p a c k i n g of c h a i n s i n hemoglobin diagram p-oxo-bis  (porphyriniron (III))  in  InTPPrCl  -viii-  -ix-  Figure  Title  Page  6.12  Differential .attentuation coefficients G 2(0 for rhombic quadrupole interaction in polycrystalline sources for intermediate state spins 1=2 and 1=5/2  107  6.13  Time-integrated attentuation coefficients G22(°°) for rhombic quadrupole interaction in polycrystalline sources for spins 1=2 and 1=5/2  108  6.14  7.1  2  181 Differential anisotropy A(t) of the Ta y-y directional correlation measured with a polycrystalline Hf-metal source Anisotropy of correlated gamma-ray emission from (a) free C d in solution (b) C d in the presence of native carbonic anhydrase (c) Hl Cd+ in the presence of apo-carbonic anhydrase m  +  m  ] QO,  112  +  m  7.2  In vivo angular correlations of gamma-rays following intravenous injection of three labelled indium compounds into mice  115  7.3  Time-differential perturbed angular correlation spectra for samples of polyglutamic acid at pH=4.0 and pH=7.8  117  8.1  Visible Absorption spectrum of protoporphyrin IX in formic acid  8.2  Visible absorption spectrum of meso-protoporphyrin IX in formic acid  122  8.3  Tic results for indium meso-protoporphyrin IX  124  8.4  Visible absorption spectrum of indium mesoprotoporphyrin IX in formic acid  126  8.5  Visible absorption spectrum of indium mesoprotoporphyrin IX in benzene  127  8.6  A schematic drawing of a CM-52 column constructed from a 30cc plastic disposable syringe  134  8.7  A schematic diagram for a fast-slow arrangement for P.A.C. experiments  -j -yj  8.8  A simplified block diagram of the electronics used in the present time-differential perturbed angular correlation experiments  139  "121  -x-  Figure  Title  9.1  TDPAC spectrum f o r ^ InMPP-Mb i n aqueous sodium phosphate b u f f e r (pH = 7.0) w i t h g l y c e r i n e added to give a f i n a l g l y c e r i n e percentage of A l .  9.2  TDPAC spectrum f o r *^InMPP-Mb i n aqueous sodium phosphate b u f f e r (pH = 7.0) w i t h o u t g l y c e r i n e  9.3  TDPAC spectrum f o r powder form  9.A  TDPAC spectrum f o r ^ ^ I n M P P IX i n dimethylformamide  9.5  TDPAC spectrum f o r  9.6  R o t a t i o n a l c o r r e l a t i o n time f o r oxyhemoglobin as a f u n c t i o n of v i s c o s i t y  A. l  Oxygen e q u i l i b r i u m c u r v e s o f m y o g l o b i n and hemoglobin  B. l  A schematic drawing o f m e s o - t e t r a p h e n y l p o r p h i n e ( f r e e base)  B.2  A s c h e m a t i c drawing o f a p o r p h y r i n  B.3  Geometries f o r p o r p h y r i n  11  111  InMPP-Mb i n the l y o p h i l i z e d  1 1 1  InTPP  i n chloroform  diacids  diacid  -xiLIST OF TABLES Table  Title  Page  2.1  Common p o r p h y r i n s and a b b r e v i a t i o n s  7  2.2  V a r i a t i o n s of M-N d i s t a n c e s w i t h i o n i c r a d i i  13  2.3  S t e r e o c h e m i c a l parameters f o r p o r p h y r i n s and m e t a l l o p o r p h y r i n s  17  2.A  Parameters of the square-pyramidal coo r d i n a t i o n group i n s e v e r a l h i g h - s p i n  20  iron(III)  porphyrins  2.5  S t e r e o c h e m i c a l parameters of the squarepyramidal c o o r d i n a t i o n group f o r lowspin c o b a l t ( I I ) porphyrins  22  2.6  Heme geometry i n myoglobin and hemoglobin derivatives  33  4.1  C r y s t a l data and c o n d i t i o n s f o r data c o l l e c t i o n  62  4.2  F i n a l atomic  68  4.3  Rigid  4.A  D e r i v e d hydrogen atom p o s i t i o n a l and thermal parameters  70  A.5  S e l e c t e d i n t e r a t o m i c d i s t a n c e s and angles  71  A.6  S e l e c t e d planes of the p o r p h y r i n m a c r o c y c l i c skeleton  73  A.7  A c o m p i l a t i o n of m e t a l l o p o r p h y r i n s w i t h metal out-of-plane displacements  74  7.1  Anisotropies for  9.1  F i t t e d v a l u e s f o r T , n , w D and 6 f o r myoglobin r e c o n s t i t u t e d wi th - - InMPP IX.  143  9.2  R o t a t i o n a l c o r r e l a t i o n times, i , and Stokes r a d i u s (R), of myoglobin, chymotrypsin, and hemoglobins  156  9.3  R o t a t i o n a l c o r r e l a t i o n t i m e s , T of myoglobin and myoglobin r e c o n s t i t u t e d w i t h m i n M P P IX at v a r i o u s temperatures and v i s c o s i t i e s  158  9.A  Stokes r a d i u s of InMPP-Mb c a l c u l a t e d from the Debye model of r e l a x a t i o n u s i n g experimental T (P.A.C.) v a l u e s  p o s i t i o n a l and thermal parameters  group parameters  59  In compounds  studied ^n vivo  c  L  L1  c  c  111  162  -xii-  ACKNOWLEDGEMENTS  I n 1977, I j o i n e d P r o f . A. G. M a r s h a l l ' s b i o p h y s i c a l  group  i n U.B.C. where I was f i r s t i n t r o d u c e d t o t h e P e r t u r b e d A n g u l a r C o r r e l a t i o n (P.A.C.) o f gamma r a d i a t i o n t e c h n i q u e , and p o r p h y r i n chemistry.  I s h o u l d l i k e t o e x p r e s s my s i n c e r e a p p r e c i a t i o n t o  P r o f . M a r s h a l l f o r h i s c o n t i n u i n g guidance and encouragement throughout my s t a y a t U.B.C. I am p a r t i c u l a r l y i n d e b t e d t o P r o f . P.W. M a r t i n o f t h e P h y s i c s Department, U.B.C. who c a r r i e d o u t t h e P.A.C. measurements, and a l s o t o Dr. R. G. B a l l who d i d t h e X-ray a n a l y s i s o f t h e i n d i u m p o r p h y r i n complex.  A s p e c i a l thanks t o M i s s Mandy o f t h e P a t h o l o g y  Department, U.B.C. who c a r r i e d out t h e v i s c o s i t y measurements. I w i s h t o acknowledge w i t h g r a t i t u d e t h e h e l p f u l and c r i t i c a l s u g g e s t i o n s from Drs. D. C. Roe, Lewis C h o i , Mr. G. A. Louma and J . L. Smith who r e a d t h i s  thesis.  A measure o f acknowledgement must be extended t o Mr. T. D e k l e v a , R. Snoek, Ms. Roxanne L e B l a n c Lemieux, and Mrs. J . C a r r u t h e r s f o r t h e i r support and encouragement. I would l i k e t o thank Dr. A. W. A d d i s o n o f D r e x e l U n i v e r s i t y , and D r s . D. D o l p h i n , B. R. James, A. S t o r r and J . T r o t t e r o f U.B.C. f o r t h e i r h e l p f u l comments and d i s c u s s i o n s . A l t h o u g h space l i m i t a t i o n s p r o h i b i t a l i s t i n g o f a l l those who have make my s t a y an e n j o y a b l e and f r u i t f u l one i n Vancouver, t o r e c o r d my g r a t i t u t e t o them.  I wish  F i n a l l y , I would l i k e t o e x p r e s s my  a p p r e c i a t i o n t o Mrs. Anna Wong f o r t y p i n g t h i s  thesis.  -1-  CHAPTER I GENERAL INTRODUCTION The  l a s t decade has w i t n e s s e d a number o f major p u b l i c a t i o n s i n  metalloporphyrin research.  A high l e v e l of continuing a c t i v i t y i n t h i s  f i e l d stems from i n t e r e s t i n b i o l o g i c a l systems t o w h i c h these compounds are r e l a t e d . conductors  Metalloporphyrins are also studied f o r developing  ( 1 ) , a n t i - c a n c e r drugs ( 2 ) , and c a t a l y s t s ( 3 ) .  important m e t a l l o p o r p h y r i n s a r e p r i n c i p a l l y  super-  The b i o l o g i c a l l y  . i r o n p o r p h y r i n s , and these  a r e t h e p o r p h y r i n s t h a t s e r v e as t h e p r o s t h e t i c group i n s e v e r a l c l a s s e s of t h e hemoproteins (4) such as hemoglobins, m y o g l o b i n s , c a t a l a s e s , e r y t h r o c r u o r i n s , some p e r o x i d a s e s , and cytochromes.  For the present  c a s e , o n l y myoglobins and hemoglobins w i l l be c o n s i d e r e d . T h i s t h e s i s i s d i v i d e d i n t o two p a r t s .  P a r t one d e a l s w i t h t h e  stereochemistry of indium(III) meso-tetraphenylporphyrin ( I n T P P r C l ) and m e t a l l o p o r p h y r i n s i n g e n e r a l .  chloride  P a r t two d e s c r i b e s t h e d e t e r -  m i n a t i o n o f t h e r o t a t i o n a l c o r r e l a t i o n time o f r e c o n s t i t u t e d m y o g l o b i n by means o f t h e d i r e c t i o n a l gamma-gamma a n g u l a r c o r r e l a t i o n  technique.  S t r u c t u r a l studies of metalloporphyrins contribute s i g n i f i c a n t l y to  the understanding  o f t h e c h e m i c a l and p h y s i c a l p r o p e r t i e s o f p o r p h y r i n s ,  and e l u c i d a t i o n o f t h e s t r u c t u r e o f i r o n p o r p h y r i n s i n p a r t i c u l a r  carries  many b i o l o g i c a l l y s i g n i f i c a n t i m p l i c a t i o n s ; t h i s a s p e c t i s d e a l t w i t h i n d e t a i l s i n Chapter I . A c c o r d i n g t o t h e most r e c e n t p u b l i s h e d c o m p i l a t i o n ( 5 ) , t h e c r y s t a l and m o l e c u l a r s t r u c t u r e s o f more than 120 p o r p h y r i n s and m e t a l l o p o r p h y r i n s had been determined by X-ray d i f f r a c t i o n by 1976. A major reason f o r t h e  -2-  h i g h and c o n t i n u i n g i n t e r e s t i n such d e t a i l s as t h e bond l e n g t h s , m e t a l d i s p o s i t i o n , and d i s t o r t i o n s i n t h e p o r p h y r i n c o r e s t r u c t u r e o f m e t a l l o p o r p h y r i n s i s t h a t pronounced v a r i a t i o n s i n t h e s e s t r u c t u r a l  parameters  o c c u r on o x y g e n - b i n d i n g , and t h e s e v a r i a t i o n s a r e proposed t o r e l a t e d i r e c t l y t o t h e c o - o p e r a t i v e o x y g e n - b i n d i n g f u n c t i o n o f hemoglobin ( 6 ) . A l t h o u g h a t l e a s t 20 m e t a l l o p o r p h y r i n complexes e x h i b i t s t r u c t u r e s w i t h the m e t a l atom d i s p l a c e d by v a r y i n g d i s t a n c e s above t h e mean p l a n e o f t h e 4 p y r r o l e n i t r o g e n s o f t h e p o r p h y r i n s k e l e t o n , v i r t u a l l y none f u l f i l l s the u s e f u l r e q u i r e m e n t s t h a t t h e m e t a l d i s p l a c e m e n t be s i m i l a r t o t h a t i n hemoglobin, and t h a t t h e m e t a l be s t a b l e t o o x i d a t i o n .  Since indium(III)  i s o x i d a t i o n - s t a b l e i n aqueous s o l u t i o n , and has an i o n i c r a d i u s s i m i l i a r to t h a t o f i r o n ( I I ) , i t was d e c i d e d t o determine t h e c r y s t a l and m o l e c u l a r s t r u c t u r e o f an i n d i u m p o r p h y r i n complex.  The p r e s e n t r e s u l t s  suggest  t h a t r e c o n s t i t u t i o n o f m y o g l o b i n o r hemoglobin w i t h i n d i u m p o r p h y r i n s s h o u l d p r o v i d e i n f o r m a t i o n o f d i r e c t r e l e v a n c e t o t h e proposed models f o r hemoglobin  function.  The p o t e n t i a l o f p e r t u r b e d a n g u l a r c o r r e l a t i o n (P.A.C.) measurements t o y i e l d m o t i o n a l and s t r u c t u r a l i n f o r m a t i o n about b i o l o g i c a l macromolecules  has been d i s c u s s e d i n s e v e r a l p u b l i c a t i o n s ( 7 - 1 2 ) .  The  s i m p l i c i t y o f e x p e r i m e n t a l measurements, i n c o m b i n a t i o n w i t h c o n c e n t r a t i o n -12 s e n s i t i v i t y a p p r o a c h i n g 10  M enhance t h e a p p e a l o f t h e P.A.C. t e c h n i q u e .  Another a t t r a c t i v e f e a t u r e o f t h e P.A.C. t e c h n i q u e i s t h e a b i l i t y t o study b o t h t h e l i q u i d and s o l i d s t a t e s , which opens t h e p o s s i b i l i t y f o r " i n v i v o " experimentation.  D e s p i t e t h e obvious p o t e n t i a l o f such a p p l i c a t i o n s ,  r e l a t i v e l y few s t u d i e s o f t h i s type have been performed.  The r e a s o n f o r  t h i s p a u c i t y o f p u b l i c a t i o n s i s probably the l a c k of v e r s a t i l i t y i n s e l e c t i v e attachment o f r a d i o a c t i v e " r o t a t i o n a l l a b e l s , " t o s p e c i f i c s i t e s on macromolecules.  T h i s t h e s i s shows t h a t  indium-Ill  -3-  l a b e l l e d p o r p h y r i n s c a n be s e l e c t i v e l y i n c o r p o r a t e d i n t o  myoglobin.  Indium has an i s o t o p e ("'""'""'"In) t h a t i s r a d i o a c t i v e w i t h an i n i t i a l s t a t e h a l f - l i f e o f 2.8 days, and the decay p r o c e s s produces two gamma r a y s i n s u c c e s s i o n , each w i t h a c o n v e n i e n t  energy f o r d e t e c t i o n .  M a r s h a l l e t a l . , (13,14) were t h e f i r s t t o show how gamma-gamma c o i n c i d e n c e measurements f o r t h i s s o r t o f energy cascade c a n g i v e d i r e c t i n f o r m a t i o n about the c h e m i c a l bonding and m o t i o n a l f l e x i b i l i t y a t an i n d i u m - l a b e l l e d s i t e on a macromolecule.  The p r e s e n t r e s u l t s ( P a r t two)  c o n f i r m the a p p l i c a b i l i t y o f t h i s measurement t e c h n i q u e t o i n d i u m - l a b e l l e d myoglobin. The p r e s e n t r e c o n s t i t u t i o n o f i n d i u m - I I I  meso-tetraphenylporphyrin  i n t o m y o g l o b i n r e p r e s e n t s t h e f i r s t m o t i o n a l probe l o c a t e d a t t h e c e n t r a l metal a t the center o f the a c t i v e s i t e of p r o t e i n . J u s t i f i c a t i o n f o r the content of t h i s t h e s i s i s two-fold. t h e s i s proposes t h a t r e c o n s t i t u t i o n . of hemoglobin w i t h i n d i u m c o u l d p r o v i d e an i n s i g h t i n t o the mechanism o f c o - o p e r a t i v e f u n c t i o n o f hemoglobin.  Secondly,  F i r s t l y , .this  porphyrins oxygen-binding  the p r e s e n t work demonstrates t h a t t h e  P.A.C. t e c h n i q u e c o u l d p r o v i d e an a l t e r n a t i v e means o f o b t a i n i n g r o t a t i o n a l c o r r e l a t i o n times o f b i o l o g i c a l macromolecules. Hemoglobin i s p o s t u l a t e d by P e r u t z (6) t o e x i s t i n two c o n f o r m a t i o n a l forms.  The form i n w h i c h t h e c e n t r a l f i v e - c o o r d i n a t e F e ( I I ) atom i s d i s -  p l a c e d above the heme p l a n e i s known as t h e "T" ("tense") form. "R"  ("relaxed")  The o t h e r  form i s one i n w h i c h t h e c e n t r a l s i x - c o o r d i n a t e F e ( I I )  atom l i e s n e a r l y c o p l a n a r w i t h the heme p l a n e .  The heme i r o n i s f i v e -  c o o r d i n a t e i n deoxy-hemoglobin (deoxy-Hb) w h i c h i s r e s p o n s i b l e f o r t h e "T" form.  On b i n d i n g m o l e c u l a r oxygen, t h e heme i r o n becomes s i x - c o o r d i n a t e  and moves i n t o t h e heme p l a n e , and t h e oxy-Hb i s s a i d t o be i n t h e "R" form.  -4-  A c c o r d i n g t o P e r u t z , t h e t r a n s i t i o n from "T" t o "R" forms causes  protein  c o n f o r m a t i o n a l changes t h a t a r e n e c e s s a r y f o r t h e observed c o - o p e r a t i v e o x y g e n a t i o n e f f e c t i n hemoglobin.  A c r i t i c a l t e s t .for t h i s proposal  would be t o " f r e e z e " one o r more s u b u n i t s i n t o t h e "T" form, and t h e n m o n i t o r changes i n t h e k i n e t i c , of  e l e c t r o n i c , and m o t i o n a l p r o p e r t i e s  t h e r e m a i n i n g n a t i v e s u b u n i t s upon o x y g e n a t i o n .  I n oxy-Hb, n a t i v e sub-  u n i t s i n the"T" forms a r e v e r y d i f f i c u l t t o o b t a i n s i n c e F e ( I I ) b i n d s oxygen t o g i v e t h e "R" form. S i n c e t h e "T" form cannot be s t a b l y m a i n t a i n e d i n oxy-Hb u s i n g n a t i v e i r o n heme, o t h e r means such as r e c o n s t i t u t i o n o r "metal r e p l a c e ment" must be sought.  Hemoglobins have been r e c o n s t i t u t e d w i t h v a r i o u s  m e t a l l o p o r p h y r i n s c o n t a i n i n g Mn, Co, Zn, and Mg i n an o x i d a t i o n s t a t e s i m i l a r to F e ( I I ) . Mn(III).  However, Co b i n d s oxygen, and M n ( I I ) a u t o x i d i z e s t o  Both Mg and Zn may be s t a b l e t o o x i d a t i o n b u t they e x h i b i t  r e l a t i v e l y s m a l l o u t - o f - p l a n e displacements of t h e m e t a l atoms above t h e p o r p h y r i n p l a n e r e l a t i v e t o t h a t of t h e Fe atom i n n a t i v e Hb. A comparison o f t h e s t r u c t u r a l d a t a on v a r i o u s m e t a l l o p o r p h y r i n s (Table 4.7) shows t h a t t h e o u t - o f - p l a n e d i s p l a c e m e n t o f i n d i u m i n InTPP:Cl i s by f a r the most s i m i l a r t o t h a t o f Fe i n h o r s e deoxy-Hb and human deoxy-Hb. Other a t t r a c t i v e f e a t u r e s o f indium a r e t h a t I n ( I I I ) i s o x i d a t i o n s t a b l e , and does not b i n d oxygen.  The c o m b i n a t i o n o f n e a r - i d e a l I n . . . P u * d i s t a n c e ,  s t a b i l i t y t o o x i d a t i o n , and i n a b i l i t y t o b i n d oxygen suggest t h a t an indium p o r p h y r i n may be an i d e a l c a n d i d a t e f o r i n d u c i n g t h e "T" c o n f o r m a t i o n .  I f the  c u r r e n t t h e o r y o f c o - o p e r a t i v i t y by P e r u t z (6) i s c o r r e c t , i n d i u m p o r p h y r i n  Indium o u t - o f - p l a n e d i s p l a c e m e n t from t h e mean p l a n e o f t h e e n t i r e macrocycle.  -5-  complexes r e c o n s t i t u t e d i n p l a c e o f heme groups i n hemoglobin s h o u l d " f r e e z e " t h e i n d i u m - l a b e l l e d s u b u n i t s i n t o t h e d e s i r e d "T" c o n f o r m a t i o n . The second j u s t i f i c a t i o n i s t h a t P.A.C. t e c h n i q u e can be used t o o b t a i n m o t i o n a l and s t r u c t u r a l i n f o r m a t i o n about b i o l o g i c a l macromolecules. Indium has an advantage t h a t i t has an i s o t o p e ("'""'""'"In) t h a t i s s u i t a b l e f o r gamma-gamma c o i n c i d e n c e measurements.  These measurements r e q u i r e  the i n c o r p o r a t i o n o f a gamma-ray e m i t t e r i n t o t h e m o l e c u l e under s t u d y . S e v e r a l i s o t o p e s c a n be used: ^"'""'" Cd(tj =49 m i n ) , ^ Z n ( t j = 9 h ) , ^ ^ H g ( t j =43 m i n ) , m  -5  "PbCt, =68 m i n ) , and most c o n v e n i e n t  In(tj=2.8 days).  -i  -3  I t i s obvious t h a t  I n i s the  i s o t o p e from-the p r a c t i c a l v i e w p o i n t .  The p r e s e n t r e s u l t s e f f e c t i v e l y demonstrate  that motional properties  such as t h e r o t a t i o n a l c o r r e l a t i o n time o f m y o g l o b i n c a n be determined by means o f t h e P.A.C. t e c h n i q u e .  T h i s work c a n be extended t o o t h e r  b i o l o g i c a l macromolecules such hemoglobins, and cytochromes. I t i s i n t e r e s t i n g t o note t h a t f o r t h e p a s t decades, many i n v e s t i g a t o r s have u t i l i z e d v a r i o u s marker substances  ranging  from p o r p h y r i n  complexes, t e t r a c y c l i n e d e r i v a t i v e s , r a d i o a c t i v e i s o t o p e s , and o t h e r substances  t o i d e n t i f y and d e l i n e a t e m a l i g n a n t  coworkers have observed tumors.  tissues.  Schwartz and  a high concentration of c e r t a i n porphyrins i n  I t i s c o n c e i v a b l e t h a t "'""'""'"In-labelled p o r p h y r i n s c o u l d be used  for tumor-localisation. (a c o n v e n i e n t  I n d i u m - I l l has i d e a l p h y s i c a l c h a r a c t e r i s t i c s  2.8 days h a l f - l i f e and gamma r a y s i n t h e d e s i r e d energy  range) f o r tumor s c a n n i n g .  -6-  PART ONE  CRYSTAL AND MOLECULAR STRUCTURE OF a , 3 , Y , < 5 TETRAPHENYLPORPHINATOINDIUM(III) CHLORIDE  -7-  CHAPTER I I  2.1  Metalloporphyrin The p o r p h y r i n s  Stereochemistry  a r e compounds formed by adding s u b s t i t u e n t s t o  the n u c l e u s o f p o r p h i n e ( F i g u r e 2.1). The n a t u r a l l y porphyrins  occurring  a r e g e n e r a l l y formed by a d d i n g s u b s t i t u e n t s t o p o s i t i o n s  1-8 and a r e named a c c o r d i n g  t o t h e number and t y p e o f s u b s t i t u e n t s .  T a b l e 2.1 l i s t s s e v e r a l p o r p h y r i n s , b o t h n a t u r a l and s y n t h e t i c w i t h t h e i r t r i v i a l names t h a t most f r e q u e n t l y employed f o r s t r u c t u r a l studies. TABLE 2.1 Common P o r p h y r i n s  and A b b r e v i a t i o n s  . . . a P o s i t i o n on r i n g Porphyrin  Protoporphyrin 2,4-Diacetyldeuteroporphyrin Mesoporphyrin^ Etioporphyrin I E t i o p o r p h y r i n 11^ Octaethylporphyring Tetraphenylporphyrin Tetra(4-pyridyl)porphyrin c  e  n  1  2  3  4  5  6  7  8 a,3,6,y  M  V  M  V  M  P  P  M.  H  M M M M E H H  Ac E E E E H H  M M M E E H H  Ac E E M E H H  M M M M E H H  P P E E E H H  P P M E E H H  M M E M E H H  H H H H H 6H C H N  C  5  5  4  1  Ac,COCH ;E,CH CH ;M,CH ;P,CH CH COOH;V,CHCH 3  2  H Proto. H OEP. 2  3  H Deut.  2  g  3  H MesoP.  2  h  H TPP. 2  2  2  2  "H Etio I. 2  2  H Etio II. 2  H TPyP. 2  The n o t a t i o n t h a t w i l l be used throughout t h i s c h a p t e r i s i l l u s t r a t e d  -8-  i n F i g u r e 2.1.  and  f o r a- and b-carbon atoms o f a p y r r o l e r i n g ,  N f o r p y r r o l e n i t r o g e n , and Cm f o r methine carbon.  Ct denotes t h e  c e n t e r o f t h e p o r p h y r i n , M a c o o r d i n a t e d m e t a l i o n , and Ne t h e c o ordinated a x i a l nitrogeneous  base.  M-N i s used t o r e p r e s e n t a m e t a l -  p y r r o l e n i t r o g e n bond d i s t a n c e , and M-Ne t h e m e t a l - a x i a l n i t r o g e n bond distance.  A non-bonded s e p a r a t i o n between two atoms i s denoted by  t h r e e c e n t e r e d d o t s ( . . . ) ; a bonded d i s t a n c e i s d e s i g n a t e d by a d a s h ( - ) . The c h i e f impediment t o t h e e l u c i d a t i o n o f m e t a l l o p o r p h y r i n s t r u c t u r e i s the d i f f i c u l t y  ( e s p e c i a l l y f o r d e r i v a t i v e s of b i o l o g i c a l l y  important  p r o t o p o r p h y r i n IX) o f o b t a i n i n g t h e s i n g l e c r y s t a l s s u i t a b l e f o r X-ray d i f f r a c t i o n a n a l y s i s . The c r y s t a l s a r e o f t e n t o o s m a l l o r when l a r g e enough, a r e o f t e n s u b j e c t t o c r y s t a l d e f e c t due t o i n t e r n a l disorder.  packing  The n a t u r e o f t h e c r y s t a l d i s o r d e r may v a r y markedly b o t h i n  k i n d and degree.  I t i s o f t e n found t h a t c r y s t a l l i n e d i s o r d e r can be  so e x t e n s i v e so as t o p r e c l u d e m e a n i n g f u l  structural  investigation.  S t r u c t u r a l a n a l y s i s can o f t e n be m i s l e a d i n g i f t h e n a t u r e o f t h e c r y s t a l l i n e d e f e c t i s not i d e n t i f i e d and i t s importance a s s e s s e d .  For  example, t h e p r e s e n t c r y s t a l l o g r a p h i c a n a l y s i s o f InTPP:Cl i n d i c a t e s non-equivalence internal  o f t h e p h e n y l t i l t a n g l e s w h i c h can be e x p l a i n e d from  packing.  D e r i v a t i v e s o f t h e symmetric p o r p h y r i n s  (H^EtiOjR^OEP, H^TPP, and  R^TPyP) a r e most f r e q u e n t l y employed f o r s t r u c t u r a l d e t e r m i n a t i o n because of t h e i r a v a i l a b i l i t y and ease i n o b t a i n i n g t h e n e c e s s a r y  single crystals.  W i t h i n the accuracy of the experimental data, there a r e only small d i f f e r e n c e s between t h e n a t u r a l l y o c c u r r i n g p o r p h y r i n s and s y n t h e t i c d e r i v a t i v e s , i n cases where s t r u c t u r e s of. b o t h have been Removal o f t h e two p y r r o l e p r o t o n s y i e l d s a p o r p h y r i n  determined. dianion that  -9-  R  5  Re  R4  F i g u r e 2.1. The nucleus of porphine, Illustrating the positions of possible s u b s t i t u e n t s and the notation used for the unique atoms of the core.  -10-  behaves as a t e t r a d e n t a t e l i g a n d w h i c h r e a d i l y complexes any o f a v a r i e t y of metal i o n s . As expected, is four.  the minimum c o o r d i n a t i o n number  A d d i t i o n of a x i a l l i g a n d s , e i t h e r n e u t r a l or a n i o n i c , g i v e s  m e t a l l o p o r p h y r i n d e r i v a t i v e s w h e r e i n the m e t a l i o n has a c o o r d i n a t i o n of f i v e , s i x or e i g h t .  Only the f i v e - c o o r d i n a t e m e t a l l o p o r p h y r i n s  a r e of i n t e r e s t i n the p r e s e n t case. t a b l e of  F i g u r e 2.2  g i v e s the " p e r i o d i c -  metalloporphyrins".  P o r p h y r i n s , i n common w i t h o t h e r m a c r o c y c l i c l i g a n d s , have a c e n t r a l hole of e s s e n t i a l l y f i x e d dimensions.  The p o r p h i n a t o  r a d i a l e x p a n s i o n and c o n t r a c t i o n i n e q u a t o r i a l p l a n e .  c o r e r e s i s t s undue In c e r t a i n metal  complexes, the m e t a l i s u n a b l e to f i t i n t o t h i s h o l e and, as has been shown by Hoard ( 1 5 ) .  Thus, the p e r p e n d i c u l a r d i s p l a c e m e n t  o f the m e t a l  atom i s r e l a t e d to the e f f e c t i v e r a d i u s , s i n c e the s i z e of the c e n t r a l h o l e i n the p o r p h y r i n i s r e l a t i v e l y c o n s t a n t .  An i n s p e c t i o n o f T a b l e  4.7  shows no s i m p l e c o r r e l a t i o n between i o n i c , r a d i i and m e t a l i o n d i s p l a c e ments.  The p o r p h y r i n s a r e not r i g i d but r a t h e r remarkably  flexible  m a c r o c y c l e m o l e c u l e s (16,17,18). I n 1961  R.J.P. W i l l i a m s  (19) p o i n t e d out t h a t the t r a n s i t i o n from h i g h  to low s p i n s h o u l d be accompanied by a marked r e d u c t i o n i n the r a d i u s of the i r o n .  ionic  He f u r t h e r suggested t h a t change i n the s t r u c t u r e  of hemoglobin on o x y g e n a t i o n  " i s r e l a t e d t o the change i n the i m i d a z o l e -  i r o n d i s t a n c e or p o s s i b l y t o the concomitant change i n the s t e r i c  arrange-  ment of the i m i d a z o l e (of the heme-linked h i s t i d i n e ) r e l a t i v e t o the porphyrin  plane".  From the c r y s t a l l o g r a p h i c s t u d i e s of m e t a l l o p o r p h y r i n s  (especially  f e r r i c p o r p h y r i n c o m p l e x e s ) , J . L. Hoard and a s s o c i a t e s demonstrated  B  Li  Al  Si  P  Zn»  Ga  Ge  As  Ag  Cd  In  Sn  Sb  Au  Hg  Tl  Pb  Bi  Na  Mg»  K  Ca  Sc  Ti  V  Cr  Mn'  Fe*  Co*  Ni*  Cu»  Rb  Sr  Y  Zr  Nb  Mo  Tc  Ru  Rh  Pd  Cs  Ba  La  Hf  Ta  W  Re  Os  Ir  Pt  ..Pr..Eu..Yb..  Th  Figure 2.2. The periodic table of metalloporphyrins.(Metals which are inserted by nature are marked with an a s t e r i s k . )  -12-  experimentally  that the h i g h  t o low  a marked r e d u c t i o n i n r a d i u s of the that the  same s h o u l d  that the  hold  s p i n t r a n s i t i o n i s accompanied iron  (15,20,21,22,23).  t r u e f o r f e r r o u s complexes.,  : h i g h - s p i n f e r r o u s i o n i n d e o x y - H b and  coordinated  and  He  predicted  Hoard then proposed  deoxy-Mb i s  i s d i s p l a c e d from the p o r p h y r i n plane  by  five-  toward the  proximal  o  h i s t i d i n e by  0.5-0.8A.  Upon b i n d i n g a n  becomes s i x - c o o r d i n a t e and porphyrin.  This  H e n d r i c k s o n and and  very  l o w - s p i n , and  p l a n a r i t y has L o v e ( 2 4 ) , and  ( 1 9 ) , and  Hoard  histidine.  S.Phillips  (26).  T h i s movement i s i n t u r n r e s p o n s i b l e f o r t h e  proposed the  m y o g l o b i n upon  stereochemical  phenomenon ( S e e A p p e n d i x A ) . a s s u m p t i o n , b a s e d on  earlier  At  ideas of Hoard  i n hemoglobin the h i g h - s p i n to low-spin w i t h a movement o f t h e  deoxy-Hb,  transition  Perutz  is  the  Williams  (19)  should  i r o n toward the. p o r p h y r i n p l a n e  con-  co-operative  of t h i s proposal (.27)., and  the  oxygenation.  mechanism f o r the  the.heart  the  i m i d a z o l e group of  B a s e d o n h i s s t r u c t u r a l s t u d i e s o f o x y ( m e t ) - and (28,29) has  by  s u g g e s t e d t h a t t h e movement o f  f o r m a t i o n a l c h a n g e s i n h e m o g l o b i n and  of  by .Huber e t a l , , ( 2 5 ) ,  i r o n atom c a u s e s a change i n t h e p o s i t i o n of the proximal  atom  i n cyanomethemoglobin  CO-erythrocuorin  (27)  iron  moves b a c k t o w a r d t h e p l a n e  been v e r i f i e d  r e c e n t l y f o r o x y - m y o g l o b i n by  Williams  oxygen molecule,, the  be  that  associated  (22,23,27,28,30, 0  31).  The  necessary the  resulting  motion of the p r o x i m a l  conformational  histidine  (>0.5A) c a u s e s  the  changes i n the p r o t e i n t h a t a r e r e s p o n s i b l e f o r  co-operativity. Obviously,  test plane,  the p r i n c i p a l m e t a l l o p o r p h y r i n  i n t e r e s t are C t . . . M , and  thus the displacement t h e M-N  bond  s t r u c t u r a l parameters of grea-  o f t h e m e t a l a t o m f r o m t h e mean  distances.  -13-  The  bond d i s t a n c e s w i t h i n the p o r p h i n e s k e l e t o n i t s e l f a r e  invariant with  respect  to the p o r p h y r i n  the s m a l l d i s t a n c e v a r i a t i o n s are due  compounds s t u d i e d .  As  to s u b s t i t u e n t e f f e c t s .  quite expected . The  M-N  bond d i s t a n c e , however, does v a r y a p p r e c i a b l y depending on the n a t u r e of o  the metal atoms c o o r d i n a t e d  to the p o r p h y r i n .  M-N  can range from 2.32A  o  i n bismuth p o r p h y r i n s  to 1.95A  i n n i c k e l porphyrins.  be expected i f the m e t a l i s p o s i t i o n e d out n i t r o g e n atoms. T a b l e i o n i c r a d i i and M-N  2.2  A l o n g M-N  simple  c o r r e l a t i o n between  distances.  V a r i a t i o n s of M-N  Metal  2.2  distances with  Ionic radius  ionic  radii  M-N  distance  o  0.64A 0.68 0.72 0.74 0.81 0.86 0.95 0.96  Fe(III) Ni(II) Cu(II) Zn(II) In(III) Pd(II) Tl(III) Bi(III)  porphinato  can  of the mean p l a n e of the p y r r o l e  shows t h a t t h e r e i s no  TABLE  The  bond  core  i s o f t e n not  o  2.07A 1.96 1.98 2.05 2.16 2.00 2.21 2.32  p l a n a r but r a t h e r e a s i l y  subjected  to d e f o r m a t i o n normal to the mean p l a n e i n t o a r u f f l e d or domed c o n f i g u r a t i o n .  -14-  T h l s o b s e r v a t i o n has been c o n f i r m e d tetraphenylporphine The  (32) and  by s t r u c t u r a l d e t e r m i n a t i o n  i t s copper and p a l l a d i u m d e r i v a t i v e s ( 3 3 ) .  I n T P P r C l a l s o e x h i b i t s marked d e v i a t i o n s from p l a n a r i t y of  porphinato  core  are necessary  of  (See Chapter IV, S e c t i o n 4.3).  Planar  the  conformations  t o ensure maximum i r - i n t e r a c t i o n between t h e s u b s t i t u e n t s  and t h e e x t e n s i v e c o n j u g a t e d  p o r p h y r i n systems.  Nonplanar  however, predominate i n c r y s t a l l i n e m e t a l l o p o r p h y r i n s . p l a n a t i o n i s that nonplanar conformations  The  conformations, likely  ex-  r e s u l t from p a c k i n g c o n s t r a i n t s  i n the c r y s t a l o r i n t r a m o l e c u l a r r e p u l s i o n s of a x i a l l i g a n d s w i t h atoms of t h e  core.  Hoard (34) examined the c o r r e l a t i o n between M-N of the m a c r o c y c l e i n m e t a l l o p o r p h y r i n s . a r e more l i k e l y  d i s t a n c e s and  He c o n c l u d e s  that  f o r c o n t r a c t e d c o r e w h i c h has s h o r t M-N  s t u d i e s (35,36) c o n f i r m Hoard's o b s e r v a t i o n s .  geometry  deformations  distances.  Recent  C u l l e n and co-workers  (35)  compared two c r y s t a l l i n e forms of NiOEP so.as to e l i m i n a t e the e f f e c t s of d i f f e r e n t s u b s t i t u e n t s on t h e r i n g on t h e geometry. The  t e t r a g o n a l form  o  has  the average Ni-N d i s t a n c e (1.93A), the s h o r t e s t M-N  p o r t e d , f o r a m e t a l l o p o r p h y r i n , and  d i s t a n c e yet r e -  the m a c r o c y c l e i s h i g h l y d i s t o r t e d  I n c o n t r a s t , the t r i c l i n i c . N i O E P . i s e s s e n t i a l l y p l a n a r and the M-N o  i s 1.96A.  (37).  distance  o  The M-N  d i s t a n c e o f 2.01A  appears t o be a n e a r l y o p t i m a l  value  of m i n i m a l s t r a i n and u n d i s t o r t e d accomodation of t h e m e t a l atom w i t h i n the c e n t r a l porphinato  core  (21).  S t r u c t u r a l s t u d i e s a l s o show t h a t f o r  a h i g h l y expanded c o r e , the most e f f i c i e n t n o n p l a n a r c o n f o r m a t i o n  i s doming  of the c o r e  this  observation.  (18,34).  The  present  study of I n T P P r C l a l s o c o n f i r m s  I n T P P r C l has a h i g h l y expanded p o r p h i n a t o  core w i t h  (M-N)^  o  d i s t a n c e of 2.16A.  As expected  the p l a n e of the f o u r p y r r o l e n i t r o g e n s i s  -15-  d i s p l a c e d i . e . domed upward by 0.1A from t h e mean p l a n e o f t h e p o r p h i n a t o core. The  f i v e - c o o r d i n a t e geometry i s a v e r y common one i n m e t a l l o p o r p h y r i n s ,  e s p e c i a l l y prominent f o r z i n c , magnesium, c o b a l t ( I I ) , and h i g h - s p i n i r o n porphyrins.  F i g u r e 2.3 r e p r e s e n t s t h e schematic  diagram o f t h e square-  p y r a m i d a l c o o r d i n a t i o n group f o r f i v e - c o o r d i n a t e m e t a l l o p o r p h y r i n s .  A  b r i e f c o n s i d e r a t i o n o f t h e P y t h a g o r e a n theorem demonstrates t h a t t h e o u t df-plane displacement,  Ct...M o f t h e m e t a l atom from t h e mean p o r p h i n a t o  core can be c a l c u l a t e d i f Ct...N and M-N d i s t a n c e s a r e known.  A modest  doming o f t h e c o r e i s a n e a r l y i n v a r i a b l e f e a t u r e among f i v e - c o o r d i n a t e m e t a l l o p o r p h y r i n s e s p e c i a l l y those w i t h l a r g e c e n t r a l m e t a l atoms. Cons e q u e n t l y , t h e doming o f t h e c o r e i n e q u a l i t y o f M...P„ < M...P . N c  toward  t h e m e t a l atom l e a d s t o t h e  G e n e r a l l y P ...P N >T  (doming) i s s m a l l c °  o  <0.05A.  F o r t h i s c l a s s o f m e t a l l o p o r p h y r i n s , t y p i c a l Ct...N v a l u e s a r e o  found i n t h e range o f 1.98-2.05A, and t h e M-N d i s t a n c e s a r e u s u a l l y o  <2.01A ( T a b l e 2.3 and 4.7).  The d i s p l a c e m e n t  o f t h e m e t a l atom from  the mean p l a n e i s a measurable p r o p e r t y o f a l l m e t a l l o p o r p h y r i n s o f o  this class.  o  The magnitude v a r i e s from 0.10A t o >0.50A.  The l e n g t h  of t h e a x i a l l i g a n d - m e t a l bond i s dependent on t h e n a t u r e o f t h e a x i a l ligand. Due t o t h e doming e f f e c t , s t e r i c i n t e r a c t i o n between t h e a x i a l l i g a n d and t h e p o r p h i n a t o c o r e i s reduced and l e s s i m p o r t a n t  i n five-  c o o r d i n a t e than s i x - c o o r d i n a t e m e t a l l o p o r p h y r i n s ; i t s importance i s o b v i o u s l y d i m i n i s h e d w i t h an i n c r e a s i n g d i s p l a c e m e n t  o f t h e m e t a l atom  out-of-plane. A short d i s c u s s i o n of the stereochemistry of i r o n porphyrins i s  -16-  Figure 2.3. A diagram of the s q u a r e - p y r a m i d a l coordination group for f i v e - c o o r d i n a t e metalloporphyrins  -17-  warranted i n view of t h e i r r e l a t i o n s h i p s to b i o l o g i c a l l y m o l e c u l e s such as hemoglobin and m y o g l o b i n .  important  Cobalt porphyrins  a l s o be i n c l u d e d because o f t h e r e l a t i o n o f c o b a l t p o r p h y r i n chemistry  will stereo-  t o t h e c o b a l t - s u b s t i t u t e d hemoproteins.  A number o f i n t e r e s t i n g f e a t u r e s emerge from t h e s t e r e o c h e m i c a l d a t a i n T a b l e 2.3.  The m e t a l atom l i e s i n p l a n e w i t h t h e f o u r p y r r o l e o  n i t r o g e n atoms o n l y i f M-N < 2.01A.  The normal r a d i u s o f t h e c e n t r a l  " h o l e " i n an u n d i s t o r t e d m e t a l l o p o r p h y r i n  has been e s t i m a t e d  o  2.01A ( 2 1 ) .  t o be o  I t i s i n t e r e s t i n g t o note t h a t . t h e v a l u e o f 2.01A c o r r e s o  ponds n e a r l y t o t h e mean o f t h e s m a l l e s t  (1.93A f o r t e t r a g o n a l N10EP) and  o  the l a r g e s t (2.21A f o r T l ( O E P ) C l ) for metalloporphyrins.  M-N v a l u e s . ( S e e  Table. 4.7) so f a r r e p o r t e d  The T a b l e 2.3 a l s o shows t h a t t h e m e t a l i o n d i s -  placement i s s u b s t a n t i a l i n each of t h e i r o n p o r p h y r i n s ; a l l o f t h e s e ( i n c l u d i n g f e r r i m y o g l o b i n ) a r e h i g h - s p i n Fe  d e r i v a t i v e s with the  e x c e p t i o n o f 2-MeImFe(II)TPP i n w h i c h i r o n atom i s i n o x i d a t i o n s t a t e two.  Stereochemical NiEtio NiDeut CuTPP PdTPP H20ZnTPP ClFeTPP Chlorohemin MeOFeMeso Porphine tet-TPP tr-TPP Fe(III)Mb  „ TABLE 2.3 n  parameters f o r p o r p ^ r i n s ^ a n d M-N 1.957 1.960 1.981 2.009 >2.05 2.049 2.062 2.073 a  -  -1.9  Ct-N 1.957. 1.960 1.981 2.009 2.042 2.012 2.008 2.022 2.051 2.054 2.065 -1.9 b  metalloporphyrins A°  >0.19 0.383 0.475 0.455  -  -0.30  Axial.Ligands None None None None H 0 a t < 2.21 A C l a t 2.19 A C l a t 2.218 A OMe a t 1.842 A None None None N a t -1.9 H0 ~2.1 A 0  2  o  0  2  Metal-nitrogen distance Radius of c e n t r a l h o l e : see t e x t O u t - o f - p l a n e d i s p l a c e m e n t o f t h e m e t a l atom From J . L. Hoard ( R e f . 22)  (distances Ref 38 39 33 33 20 40 41 23 42 17 43 44  -18-  2.1a  Iron  Porphyrins  I r o n p o r p h y r i n s p r o v i d e the t y p i c a l examples of t h e i n f l u e n c e of the s p i n - s t a t e s and o x i d a t i o n s t a t e s o f the c e n t r a l m e t a l i o n s on t h e stereochemistry  of metalloporphyrin.  The e f f e c t i v e r a d i u s o f h i g h - s p i n  i r o n ( I I I ) i o n i s too l a r g e t o a l l o w the i r o n t o f i t i n t o t h e c e n t r a l h o l e . o  A t y p i c a l displacement expected.  o f the i r o n o f about 0.48A o u t - o f - p l a n e  A s m a l l amount of doming of the p o r p h i n a t o  i s as  c o r e toward t h e i r o n ( I I )  o  i o n i s f r e q u e n t l y observed  with P . . P c >  N  < 0.05A.  A qualitative picture  f o r t h i s o b s e r v a t i o n i s gained by c o n s i d e r i n g a square-pyramidal for a high-spin iron  model  porphyrin.  In the simple model, the Z - d i r e c t i o n i s d e f i n e d as normal to t h e porphinato  core  ( F i g u r e 2.4).  The d: 2 o r b i t a l of a coordinated x y metal i o n i s then i n a p l a n e p a r a l l e l t o the mean p o r p h i n a t o c o r e . A m e t a l i o n where the d 2_ 2 o r b i t a l i s populated w i l l be r e l a t i v e l y l a r g e x y and have commensurately long M-N bonds. metalloporphyrin, and  t h e r e i s one u n p a i r e d  I n any h i g h - s p i n F e ( I I ) or F e ( I I I ) e l e c t r o n i n each o f the 3 d ^ y2 2  3 d 2 o r b i t a l s i n the metal atom. The presence o f the u n p a i r e d e l e c t r o n z  i n 3d^2_y2 o r b i t a l i s r e s p o n s i b l e f o r t h e s u b s t a n t i a l displacement i r o n from t h e plane o f the p y r r o l e n i t r o g e n atoms.  In a low-spin  o f the Fe(II)  or F e ( I I I ) , the p a i r i n g of e l e c t r o n s p i n s i n the d ', d, , and d „ v a l e n c e xy yz xz 6  r  s h e l l o r b i t a l s o f the m e t a l atom a l l o w s f u l l u t i l i z a t i o n o f t h e unoccupied 3d^2_y2 d ^ 2 b i t a l s f o r complexing two a x i a l l i g a n d s . The h i g h - s p i n complex o f 2 - m e t h y l i m i d a z o l e i r o n ( I I ) (45) i s a f i v e a  n  3<  o r  z  c o o r d i n a t e , square-pyramidal,  o  and the i r o n ( I I ) i s d i s p l a c e d 0.42A from o  the mean plane o f the f o u r p y r r o l e n i t r o g e n s and 0.55A from the mean o  porphinato  core.  The doming i . e . P^...P  s e p a r a t i o n i s 0.13A.  -19-  Figure 2 . 4 . E n e r g y - l e v e l diagram square-pyramidal coordination.  for  -20-  Both t h e F e ( I I ) and F e ( I I I ) c a n form l o w - s p i n complexes when c o o r d i n a t e d t o two a p p r o p r i a t e type of a x i a l l i g a n d s .  According to  Hoard's o b s e r v a t i o n (15,20,21,23) i n t h e l o w - s p i n s i x - c o o r d i n a t e complexes, t h e i r o n i s c o p l a n a r o r n e a r l y e o p l a n a r w i t h t h e p o r p h i n a t o core.  A s u b s t a n t i a l l y s h o r t e r Fe-N bond d i s t a n c e r e l a t i v e t o t h e h i g h -  s p i n can a l s o be a n t i c i p a t e d . The s t r u c t u r e of a l o w - s p i n s i x - c o o r d i n a t e ( I n ^ F e T P P ^ C l  has shown  o  t h a t t h e p o s i t i o n o f t h e i r o n ( I I I ) i s o n l y 0.009A (30) from t h e mean o  porphinato n i t r o g e n s .  The (Fe-N). d i s t a n c e o f 1.989A i s s i g n i f i c a n t l y av o  s h o r t e r than those o f t h e h i g h - s p i n complexes w i t h ( F e - N )  a v  > 2.04A (See  T a b l e 2.4). TABLE 2.4 Parameters o f t h e Square-Pyramidal C o o r d i n a t i o n Group i n Several High-Spin I r o n ( I I I ) Porphyrins  D i s t a n c e (A) Derivative  (Fe..N)  Ct...N  Ct...Fe  Fe-X  2.062(10)  2,008  0.475.  2.218(6)  41  2.073(6)  2.022  0.455  1.842(4)  23  2.049(9)  2.012  0.38  2.192(12)  40  2.087(5)  2.027  0.50  1.763(1)  48  (SCN)FeTPP  2.065(5)  2.007  0.485  1.957(5)  49  N^FeTPP  2.055  2.027  0.34  1.909  50  1.73  51  1.752(1)  52  av ClFeProtoDME  3  CH OFeMesoPDME  b  3  ClFeTPP 0(FeTPP)  2  0(FeProtoDME) 0(FeODM)  c 2  a 2  -  2.08 2.065(8)  2.002  0.53  ^ Dimethyl ester of protoporphyrin Dimethyl e s t e r of mesoporphyrin a,Y-Dimethyloctaethylporphyrin From S c h e i d t ( R e f . 4 7 ) .  Ref  -21-  The s t e r e o c h e m i c a l p a t t e r n i s m a i n t a i n e d d e r i v a t i v e i n w h i c h two n o n - e q u i v a l e n t  f o r an F e ( I I I ) p o r p h y r i n  a x i a l ligands are coordinated. o  I n N ( P y ) F e T P P ( 4 6 ) , t h e F e ( I I I ) i o n i s c e n t e r e d t o w i t h i n 0.03A and has 3  o  a shortened  (Fe-N) d i s t a n c e o f 1.990A. av  The l o w - s p i n F e ( I I ) p o r p h y r i n s e x h i b i t s t e r e o c h e m i c a l similar to Fe(III) derivatives.  The F e ( I I ) i o n i n t h e  Pip„FeTPP (31) i s e x a c t l y c e n t e r e d . ^  centrosymmetric  The e q u a t o r i a l (Fe-N)  o  behavior  av  bond  d i s t a n c e i s 2.004A w h i c h i s s l i g h t l y l a r g e r than t h e c h a r a c t e r i s t i c l o w spin Fe(III) derivatives. 2.1b  Cobalt  Porphyrins  A l l c o b a l t p o r p h y r i n s , e i t h e r c o b a l t ( I I I ) , d^, o r c o b a l t ( I I ) , d^, a r e l o w - s p i n complexes.  U n l i k e i r o n p o r p h y r i n s , c o b a l t complexes do not  exhibit spin-state transitions.  The s t e r e o c h e m i s t r y o f c o b a l t p o r p h y r i n s  i s g r e a t l y i n f l u e n c e d by t h e o x i d a t i o n s t a t e s and t h e number o f a x i a l ligands. T a b l e 2.5 g i v e s t h e s t e r e o c h e m i c a l parameters o f f i v e - c o o r d i n a t e l o w - s p i n c o b a l t ( I I ) p o r p h y r i n d e r i v a t i v e s . T a b l e 2.3 shows t h a t d i s p l a c e ment o f t h e m e t a l atom from t h e mean p o r p h i n a t o n i t r o g e n p l a n e o c c u r s  only  o  i f t h e complexing M-N > 2.01A. An i n s p e c t i o n o f T a b l e 2.5,. however, i n d i c a t e s t h a t c o b a l t p o r p h y r i n s do not obey t h e above o b s e r v a t i o n ; t h e c o b a l t atom i s d i s p l a c e d from t h e mean.plane o f t h e p y r r o l e n i t r o g e n s by the same d i s t a n c e s i r r e s p e c t i v e whether t h e M-N bond, d i s t a n c e i s g r e a t e r o  o r l e s s than 2.01A.  W i t h an unoccupied  d^2_y2 o r b i t a l , t h e c o b a l t ( I I )  i s a p p r o p r i a t e l y found t o have a s m a l l e r o u t - o f - p l a n e d i s p l a c e m e n t high-spin iron porphyrins.  than  -22-  TABLE 2.5 S t e r e o c h e m i c a l Parameters of t h e Square-Pyramidal  C o o r d i n a t i o n Group f o r  Low-Spin C o b a l t ( I I ) P o r p h y r i n s  Distance Derivative  o  (A)  (Co-N) av  Co-N  NMeimCoTPP  1,.977(3)  2.•157(3)  0...13  NMelmCoOEP  1..96(1)  2..15(1)  (3,5-Lut)CoTPP  2,.000(5)  DiMeImCoTPP  1,.985(2)  N  b  Co...P„ N  ...P ax c  Ref.  0.14  2..30  54  0..13  0.16  2,.33  55  2.,161(5)  0..14  0.17  2..33  56  2.,216(2)  0..15  0.18  2..37  57  a  ax  Co...P  N c  i s t h e n i t r o g e n atom of t h e a x i a l l i g a n d  ax  DiMelm i s 1 , 2 - d i m e t h y l i m i d a z o l e  From S c h e i d t ( R e f . 47)  The P i p C o ( I I I ) T P P 2  +  (Co,d ) c a t i o n (53) and l o w - s p i n 4  4 (Fe,d ) a r e b o t h i s o e l e c t r o n i c and i s o s t r u c t u r a l .  Pip Fe(II)TPP 2  Both p o r p h i n a t o  cores  o  are e s s e n t i a l l y p l a n a r .  The ( C o - N ) ^ bond d i s t a n c e i s 1.978A compared t o  o  the(Fe-N)  av  o f 2.004A.  The d i f f e r e n c e between t h e two M-N d i s t a n c e s i s  the d i r e c t consequence o f t h e d i f f e r i n g n u c l e a r charge on t h e two i o n s . The p o r p h i n a t o  core o f 0 N(3,5-Lut)CoTPP (36) i s e x t r e m e l y 2  ruffled  o  w i t h the corresponding  short (Co-N)  d i s t a n c e o f 1.954A  a v  r e p r e s e n t s t h e minimum C o ( I I I ) - N d i s t a n c e . o  which  The a x i a l Co-N^.^ d i s t a n c e i s  1.948A as opposed t o t h e a x i a l Co-N,., _ . d i s t a n c e of "2.036A. (3,5-Lut) c  probably  The d i f f e r e n c e  -23-  i n t h e two bond l e n g t h s p r i m a r i l y r e f l e c t s of t h e two a x i a l  ligands.  the d i f f e r i n g s t e r i c  requirements  - 2 4 -  2.2  A p p l i c a t i o n of Metalloporphyrin Stereochemistry to Heme Stereochemistry i n Hemoproteins (Myoglobin and Hemoglobin) This s e c t i o n concerns the a p p l i c a t i o n of metalloporphyrin stereo-  chemistry discussed i n Section 2.1 to heme stereochemistry i n myoglobin and hemoglobin.  The o b j e c t i v e i s to show how s t r u c t u r a l determination of  metalloporphyrins can provide an i n s i g h t i n t o the mechanism of r e v e r s i b l e oxygenation i n hemoglobin and myoglobin.  I t must be pointed out that  there i s s t i l l a l o t of controversy and continuing debate concerning the a l l o s t e r i c model proposed by Perutz on the oxygen-binding c o - o p e r a t i v i t y phenomenon i n hemoglobin (28,58). In hemoglobin the ferrous heme i s d i r e c t l y anchored to the g l o b i n (protein) framework through the a x i a l complexing bond formed by the i r o n ( I I ) atom with an imidazole nitrogen atom of the proximal h i s t i d i n e residue (F8).  In the low-spin, oxy-Hb, molecular oxygen occupies the  vacant s i x t h p o s i t i o n i n the coordination group of the i r o n atom. There i s no s i x t h ligand i n the high-spin, deoxy-Hb. When the s t r u c t u r e of hemoglobin was f i n a l l y solved, the hemes (59-63) were found to l i e i n i s o l a t e d pockets with entrances on the surface of the subunits.  The minimum spacing between any two heme i r o n o  atoms i n deoxy-Hb or oxy-Hb i s >25A (28,6).  D i r e c t heme-heme i n t e r a c t i o n s  hence are an implausible source of co-operative phenomenon.  As Perutz  (6) posed the question "without contact between them (hemes) how could one of them sense whether the others had combined w i t h oxygen?". Yet communication between the hemes i n each subunit i s p r e r e q u i s i t e because a l l the co-operative e f f e c t s disappear i f the hemoglobin molecule i s merely s p l i t in half.  I t was  noted by Hoard i n 1966  (27) t h a t the p r o b a b l e  starting point  f o r a s t e r e o c h e m i c a l mechanism of c o - o p e r a t i v e i n t e r a c t i o n was  the move-  o  ment >0.5A of the i r o n atom r e l a t i v e t o the p o r p h i n a t o  c o r e upon  oxygenation  accompanied by t h e concomitant h i g h - s p i n t o l o w - s p i n t r a n s i t i o n . subsequently  In Perutz's  d e v e l o p e d mechanism (28,29) f o r the c o - o p e r a t i v e o x y g e n a t i o n  of  o  hemoglobin, t h e p r i m a r y Py...Ne  t r i g g e r i s t h e s h r i n k a g e of about 0.90A i n t h e  d i s t a n c e t h a t accompanies the t r a n s i t i o n from h i g h - s p i n  five-  c o o r d i n a t i o n i n deoxy-Hb t o l o w - s p i n s i x - c o o r d i n a t i o n i n oxy-Hb. important  It is  t o n o t e t h a t o n l y t h e mean p l a n e , Py, of the e n t i r e m a c r o c y c l e  i n deoxy-Hb t h a t can be i d e n t i f i e d ; t h e p r o t o p o r p h y r i n i s viewed "edge-on as a r a t h e r t h i c k band o f u n r e s o l v e d  e l e c t r o n density i n which a s u b s t a n t i a l  doming of the p o r p h y r i n c o r e toward, t h e . i r o n atom may M-N  d i s t a n c e i n both  (non-equivalent)  a- and  be w h o l l y  obscured".  3 - s u b u n i t s of deoxy-Hb a r e  o  estimated  t o be ^0.6A.  According  to P e r u t z ' s p r o p o s a l , t h e r e must be  " t e n s i o n " i n t h e a x i a l c o n n e c t i o n between the ferroheme and (protein).  a  the g l o b i n  A comparison of the Py...Ne d i s t a n c e i n deoxy-Hb w i t h  an  e x t e r n a l l y u n c o n s t r a i n e d , f i v e - c o o r d i n a t e . 2-MeImFe(II)TPP (64,65) p r o v i d e s d i r e c t evidence  f o r Perutz's proposal.  s t e r i c a l l y hindered  I t i s i n t e r e s t i n g to note that  2-Methyl i m i d a z o l e r a t h e r than u n s u b s t i t u t e d  i s used as -the a x i a l l i g a n d i n the h i g h - s p i n  imidazole  five-coordinate species.  The  r e a s o n i s t o p r e c l u d e t h e f o r m a t i o n of the l o w - s p i n s i x - c o o r d i n a t e s p e c i e s .  Py...P£ i s the p e r p e n d i c u l a r d i s t a n c e s e p a r a t i n g t h e a x i a l n i t r o g e n atom (Ne) of the heme-linked h i s t i d i n e (F8) from the mean p l a n e (Py) of the ferroheme.  -26-  Th e Pu...Ne d i s t a n c e i s 2.90A i n deoxy-Hb as opposed t o 2.60A in  t h e u n c o n s t r a i n e d h i g h - s p i n , f i v e - c o o r d i n a t e 2-MeImFe(II)TPP ( 6 4 ) . o  T h i s c o r r e s p o n d s t o a s h o r t e n i n g o f t h e Pu...Ne by 0.30A i n 2-MeImFe(II)TPP relative  t o t h e Pu...Ne i n deoxy-Hb.  I n o t h e r words, t h e a x i a l  between t h e ferroheme and t h e g l o b i n i s i n t e n s i o n .  connection  Another p i e c e o f  e v i d e n c e comes from t h e h i g h - s p i n (l-Melm)Mn(TPP) complex ( 6 6 ) . The o  o  Mn...Pc d i s t a n c e i s 0.515A and t h e doming parameter i s s t i l l  o n l y 0.04A  o  (66)  a s compared t o Fe...Pu=0.75A i n h o r s e deoxy-Hb.  U n l e s s t h e Fe...Pu o  d i s t a n c e i n deoxy-Hb i s o v e r e s t i m a t e d by a t l e a s t 0.25A, i t appears t h a t the  a x i a l c o n n e c t i o n must be i n t e n s i o n .  Hopfield  (67) has quoted an  o  e r r o r o f ±0.1A i n c o n n e c t i o n w i t h t h e o u t - o f - p l a n e d i s p l a c e m e n t o f t h e F e ( I I ) atoms i n deoxy-Hb. Some f u r t h e r e v i d e n c e i n s u p p o r t o f t h e t e n s i o n model come from n.m.r. s t u d i e s o f normal and abnormal - hemoglobin's (68-73).  L i t t l e and  I b e r s (55) have d i s c u s s e d t h e p r o s and cons o f t h e n.m.r. and r e s o n a n c e Raman s t u d i e s . o  P e r u t z (6,62) e s t i m a t e d t h a t t h e Fe atom i s d i s p l a c e d by 0.75A from the mean p l a n e o f t h e 24-atoms p o r p h y r i n s k e l e t o n . i n h o r s e deoxy-Hb.  It  o  has been e s t i m a t e d t h a t as much as ^0.20A i s due t o doming o f t h e c o r e o  toward t h e Fe atom l e a v i n g 0.55A t o t h e o u t - o f T - p l a n e d i s p l a c e m e n t o f t h e The Pu...Ne i n deoxy-Hb i s t h e summation.of Fe...Pu=0.75A and Fe...Ne=^2.15A. The Pu...Ne i n 2-MeImFe(II)TPP i s o b t a i n e d by t h e summation o f Fe...P = 0 . 4 2 1 , P . . P = 0 . 0 5 A and Fe...Ne=2.16(Cos 10.3)A where 10.3 i s t h e a n g l e o f t i l t o f t h i s bond from t h e normal. N>  c  -27-  o  i r o n (64).  The magnitude of the doming of 0.20A can be c a l c u l a t e d  as  follows:  The s t e r e o c h e m i c a l parameters f o r 2T-MeImFe(II)TPP  a r e : Fe-Np=2.086A,  o  Fe...P =0.42, and Ct...Np=2.044A. N  I f however, the Ct...Np. d i s t a n c e i s  o  decreased t o 2.G1A  (minimum e q u a t o r i a l s t r a i n t o the m a c r o c y l e r i n g ; i n o  human deoxy-Hb, i t i s 2.0-A  (74)) w h i l e m a i n t a i n i n g the Fe-Np d i s t a n c e a t  o  2.086A, from the Pythagorean theorem, the Fe...P o  c a l c u l a t e d t o be 0.55A.  d i s p l a c e m e n t can be  o  I n o t h e r words,> Q.20A i s t o be a s s i g n e d t o doming  of t h e c o r e . As can be seen from F i g u r e 2.6, t r a n s i t i o n from deoxy-Hb t o oxy-Hb o  i n v o l v e s a s h r i n k a g e o f ^G.9A  i n the Eu...Ne w h i c h i s the sum of t h r e e  -28-  components: (a) a d e c r e a s e i n Fe-Ne a x i a l bond, (b) a movement of i r o n atom toward the Py p l a n e , and Py and P^ p l a n e s i n t o c o p l a n a r  the  (c) the movement r e q u i r e d t o b r i n g  or near c o p l a n a r .  I t i s important  to  p o i n t out the c a l c u l a t i o n i n v o l v e s bond d i s t a n c e s and d i s p l a c e m e n t s of the m e t a l atoms t a k e n from a p p r o p r i a t e m e t a l l o p o r p h y r i n s  (55),  The  s t r u c t u r e of oxy-Hb i s s t i l l a w a i t i n g ; F e ( I I ) a u t o x i d i s e s r e a d i l y t o F e ( I I I ) g i v i n g met-Hb. The  replacement of the n a t u r a l p r o s t h e t i c group i . e . i r o n  p o r p h y r i n IX w i t h d i f f e r e n t m e t a l l o p o r p h y r i n s  i n m y o g l o b i n and  has proved t o be a u s e f u l approach ( 7 5 ) . . M e t a l l o p o r p h y r i n s  proto-  hemoglobin  containing  z i n c (75,76), manganese (77-81),  copper ( 7 7 ) , and n i c k e l (82) have been  r e c o n s t i t u t e d i n t o hemoglobins.  T h e i r p r o p e r t i e s have been s t u d i e d  compared w i t h the n a t i v e hemoglobin.  These r e c o n s t i t u t e d p r o t e i n s  i n c a p a b l e of r e v e r s i b l e o x y g e n a t i o n .  Hoffman and a s s o c i a t e s  and are  (83-86)  have shown t h a t c o b a l t s u b s t i t u t e d m y o g l o b i n . (CoMb) and hemoglobin (CoHb) can combine r e v e r s i b l y and c o o p e r a t i v e l y w i t h m o l e c u l a r  oxygen. The  oxygen  a f f i n i t y i s reduced by 10-100 times f o r CoMb and.CoHb compared to n a t i v e Mb  and Hb  (86).  S i n c e l o w - s p i n C o ( I I ) e x h i b i t s a s m a l l e r . i o n i c r a d i u s than does h i g h - s p i n . F e ( I I ) , Hoffman (83-85) p r e d i c t e d the displacement, of the  Co(II)  atom i n CoHb would be r e l a t i v e l y s m a l l compared w i t h t h a t of the F e ( I I ) i n deoxy-Hb. was  The  s t r u c t u r e of the l o w - s p i n , f i v e - c o o r d i n a t e 1-MeImCo(II)TPP  s u b s e q u e n t l y determined by S c h e l d t  (54),  As p r e d i c t e d by Hoffman, o  S c h e l d t found t h a t the c o b a l t atom.is d i s p l a c e d 0.13A  from the mean p l a n e o  of the p o r p h i n a t o  n i t r o g e n s and  the doming (Py.-.P^) i s o n l y ^O.OIA; o  consequently,  the c o b a l t atom i s d i s p l a c e d by ^0.14A from the mean p l a n e  T  2.27  \ \2  he  82  0.55  •N deoxy-  1.97A  \ •N-  A=0.85A  Hb  N-  he  V i  NJ VO  I  Ne 2I15 |  Co  } 2.31  A  N  •N—Co —  deoxy-CoHb Figure of  2.6.  1.93A  A=0.38A  0  I l l u s t r a t i o n o t c a l c u l a t i o n ot t h e  t h e p r o x i m a l h i s t i d i n e in Hb(top) and Little & I b e r s  (Ref. 55)  total  movement  CoHb(bottom).The  d i s p l a c e m e n t s are t a k e n relative to the 24-atom From  N-  porphyrin c o r e .  -30-  of t h e p o r p h y r i n .  S i m i l a r l y , i n t h e l o w - s p i n , f i v e - c o o r d i n a t e 1-MeImCo(II)OEP, o  L i t t l e and I b e r s (55) found t h e Co atom i s d i s p l a c e d by 0.13A from t h e mean o  p l a n e o f t h e p y r r o l e n i t r o g e n atoms and 0.16A from t h e mean p l a n e o f t h e porphyrin core.  I t appears t h a t t h e o u t - o f - p l a n e  displacement  o f t h e Co  o  atom i n CoHb i s u n l i k e l y t o exceed 0.2A. (83),  On t h i s b a s i s , Hoffman e t a l . ,  and L i t t l e e t a l . , (55) q u e s t i o n e d whether t h e r e l a t i v e l y  out-of-plane displacement  o f t h e Co atom would b e c o n s i s t e n t w i t h t h e t r i g g e r  mechanism f o r c o o p e r a t i v e o x y g e n a t i o n The u n p a i r e d  small  proposed by P e r t u z  (28,29).  e l e c t r o n i n t h e 3d^2_y2 °tbital o f t h e h i g h - s p i n o  F e ( I I ) atom a c c o u n t s f o r t h e s u b s t a n t i a l Fe..,P  displacement  o f 0.42A  i n 2-MeImFe(II)TPP, whereas t h e absence o f such an e l e c t r o n i n t h e l o w o  s p i n C o ( I I ) atom a l l o w s t h e Co...P^ displacement  t o be 0.13A i n t h e  l-MeImCo(II)TPP (54) and 1-MeImCo(II)OEP ( 5 5 ) . I n o t h e r words, i n deoxy-CoHb, t h e Co atom i s a l r e a d y c l o s e r t o t h e mean p o r p h y r i n than Fe atom i n deoxy-Hb and i s l i k e l y t o have c r e a t e d a r e a l i n t h e Co-Ne c o n n e c t i o n .  plane  strain  T h i s i s demonstrated by t h e l e n g t h e n i n g o f o  the Co-Ne bond i n DiMelmCoTPP (Co...P =0.15A) r e l a t i v e t o 1-MeImCoTPP N o  (Co..,P^=0.13A) as shown i n T a b l e 2.5.  More s t r e t c h and enhanced t e n s i o n  i n the a x i a l connection w i l l r e q u i r e greater s t r a i n i n the g l o b i n i n deoxy-CoHb t h a n i n deoxy-Hb and c o n s e q u e n t l y s e p a r a t i o n r e a l t i v e t o t h a t i n deoxy-Hb. t h a t t h e g l o b i n can support quaternary  a r e d u c t i o n i n t h e Pu...Ne  The above argument assumes  i n c r e a s e d t e n s i o n i n Co-Ne bond so t h a t  s t r u c t u r e o f deoxy-Hb i s r e t a i n e d .  F i g u r e 2.6 a l s o i l l u s t r a t e s t h e c a l c u l a t i o n o f t h e t o t a l movement of t h e p r o x i m a l h i s t i d i n e i n Hb and CoHb by L i t t l e and I b e r s ( 5 5 ) . However, i t i s i m p o r t a n t porphinato  t o p o i n t out t h a t no doming o f t h e c o b a l t  c o r e i s assumed.  The t o t a l movement o f t h e p r o x i m a l  histidine  -31-  i s estimated  t o be 0.38A which i s a p p r o x i m a t e l y  h a l f t h a t f o r Hb.  On  t h i s b a s i s , L i t t l e and I b e r s c o n c l u d e t h a t " i t i s u n l i k e l y t h a t such a o  s m a l l movement (0.38A) c o u l d t r i g g e r t h e c o n f o r m a t i o n  changes observed  i n Co-Hb". A counterproposal  has been advanced by Hoard and S c h e i d t (64)  t h a t moderate doming o f t h e p o r p h i n a t o  c o r e i n c o m b i n a t i o n w i t h modest  s t r e t c h i n t h e Co-Ne bond can r e a d i l y l e a d t o an Py...Ne s e p a r a t i o n o a p p r o a c h i n g 2.90A i n deoxy-Hb ( F i g u r e 2.7). I n 1974, Hoard (65) proposed o o the doming t o be 0.3A i n s t e a d o f 0.5A.  The essence o f t h e Hoard and  S c h e i d t argument i s t h a t t e n s i o n w i t h i n t h e p r o t e i n i n deoxy-CoHb not o n l y causes t h e p o r p h y r i n t o undergo s u b s t a n t i a l doming b u t a l s o the C o - N e ( h i s t i d i n e ) bond. o  The doming r a i s e s t h e Co atom o  d i s p l a c e m e n t t o ^0.70A c o m p a t i b l e  increases  out-of-plane  t o t h e Fe atom (0.75A) i s h o r s e deoxy-  Hb. R e s u l t s from s e v e r a l s t u d i e s (87,88) a r e a g a i n s t t h e t e n s i o n model i n Co-Hb proposed by Hoard e t a l . , (64).  Resonance Raman s t u d i e s o f  CoHb and Hb by Woodruff e t a l . , (87) i n d i c a t e t h a t t h e C o ( I I ) atom o u t o o f - p l a n e d i s p l a c e m e n t i n deoxy-CoHb cannot exceed 0.2A.  Besides, the  resonance Raman spectrum o f deoxy-CoHb i s e s s e n t i a l l y t h e same as f o r l-MeImCo(II)OEP.  EPR experiments w i t h CoHb and model systems (88) a l s o  g i v e s no c o n v i n c i n g i n d i c a t i o n s o f t e n s i o n w i t h i n t h e g l o b i n t h a t would increase the out-of-plane  d i s p l a c e m e n t o f t h e Co atom o r s t r e t c h t h e  C o - N e ( h i s t i d i n e ) bond i n CoHb. However, t h e s t r u c t u r e o f spermwhale oxymyoglobin r e c e n t l y determined by P h i l l i p s  (26) p r o v i d e s  f u r t h e r e v i d e n c e f o r P e r u t z ' s a l l o s t e r i c model.  The X-ray d a t a was c o l l e c t e d a t -12°C t o p r e v e n t a u t o x i d a t i o n o f F e ( I I ) t o F e ( I I I ) g i v i n g met-Mb.  P h i l l i p s found t h a t t h e i r o n atom i s d i s p l a c e d  2.25 A  N. 2.15 A  i  UJ ho  0.13  •N  N:  I  0.03 CoHb  Co(1-Me-Im)(OEP) Figure  2.7.  Illustration  o f p o s s i b l e p o i n t s of  Co(II)  porphyrin prosthetic  Hoard  &  From  Scheidt(6A).  Little & I b e r s (Ref. 55)  group  d i s t o r t i o n of  the  as proposed  by  -33from t h e mean p o r p h y r i n p l a n e by 0.26A towards t h e p r o x i m a l h i s t i d i n e ( F 8 ) , and t h e bound oxygen has t h e bent-geometry.  So f a r t h e r e i s no measure  o f t h e c o r r e s p o n d i n g Fe d i s p l a c e m e n t i n oxy-Hb because under t h e normal c o n d i t i o n s o f X-ray c r y s t a l l o g r a p h y , oxy-Hb i s r e a d i l y o x i d i s e d t o met-Hb.  I t i s a common n o t i o n t h a t t h e Fe atom i s c o p l a n a r w i t h t h e heme  p l a n e i n oxy-Hb, however, t h e s t r u c t u r e o f oxy-Mb i n d i c a t e s t h i s i s n o t the c a s e . An i n t e r e s t i n g f e a t u r e o f oxy-Mb i s t h a t t h e heme-linked i m i d a z o l e o f h i s t i d i n e (F8) and Fe-0-0 a r e a p p r o x i m a t e l y c o p l a n a r and e l c i p s e d w i t h the Fe-N bond o f t h e p y r r o l e I I r i n g .  I t seems p r o b a b l e t h e s t e r i c  arrangement o f t h e h i s t i d i n e (F8) and Fe-0-0 p l a n e t h a t p r e v e n t t h e Fe atom from b e i n g c o p l a n a r w i t h t h e p o r p h y r i n p l a n e . T a b l e 2.6 shows t h a t t h e t r a n s i t i o n from deoxy-Mb t o oxy-Mb i n v o l v e s o  the movement o f t h e Fe atom towards t h e heme p l a n e and a s h r i n k a g e o f 0.59A o  i n t h e Pu...Ne as opposed t o ^0.90A i n t h e t r a n s i t i o n from deoxy-Hb t o met-Hb.  The d i f f e r e n c e i n s h o r t e n i n g of t h e Pu...Ne i s c o n c e i v a b l y  attri-  buted t o t h e f a c t t h a t m y o g l o b i n i s a s i n g l e p o l y p e p t i d e w h i c h hence does not e x h i b i t o x y g e n - b i n d i n g c o - o p e r a t i v i t y .  P h i l l i p s (26) c l e a r l y demon-  s t r a t e s t h a t t h e Fe atom does move towards t h e p o r p h y r i n on o x y g e n a t i o n as expected a c c o r d i n g t o P e r u t z ' s a l l o s t e r i c model ( 6 , 6 2 ) . TABLE 2.6  Heme geometry i n Mb and Hb d e r i v a t i v e s Deoxy-Mb  Oxy-Mb  Met-Mb  CO-Mb  Deoxy-Hb  Met-Hb  Fe-(heme p l a n e )  0.55  0.26*  0.40  0.24  0.60(a-heme) 0.07(a-heme) 0.63(8-heme) 0.21(g-heme)  Fe(Ne)-(hemeplane)  2.6  2.3  2.5  2.4  2.6(a-heme) 2.8(g-heme)  Values are given i n A * V a l u e from r e f i n e d model ( P h i l l i p s , p r i v a t e communication) Adapted from P h i l l i p s (Ref.26)  2.2(a-heme) 2.4(g-heme)  -34-  2.3  M y o g l o b i n and Hemoglobin - The Heme Group/Globin  Structure  The d e t a i l s o f t h e s t r u c t u r e o f m y o g l o b i n a r e m o s t l y d e r i v e d from t h e i m p r e s s i v e X-ray c r y s t a l l o g r a p h i c s t u d i e s o f J . C. Kendrew e t a l . , (44, 89,90), and t h e s t r u c t u r e o f hemoglobin from M. F. P e r u t z e t a l . ,  (61,62).  M y o g l o b i n o f 17,000 m o l e c u l a r w e i g h t r e p r e s e n t s t h e s i m p l e s t hemop r o t e i n capable of r e v e r s i b l e oxygenation.  T h i s p r o t e i n c o n t a i n s o n l y one  p o l y p e p t i d e c h a i n o f 153 r e s i d u e s and one heme group p e r m o l e c u l e . I n c o n t r a s t , mammalian hemoglobins have a m o l e c u l a r w e i g h t o f near 65,000 and a r e made up o f f o u r p o l y p e p t i d e c h a i n s , i d e n t i c a l i n p a i r s (a and B chains).  The a - c h a i n s have 141 amino a c i d r e s i d u e s each w h i l e t h e $-  c h a i n s have 146 r e s i d u e s each. porphyrin IX, red c o l o r .  Each c h a i n h a r b o r s  one heme ( f e r r o - p r o t o -  . F i g u r e 2.8) w h i c h when oxygenated, g i v e s b l o o d i t s  A s i n g l e p o l y p e p t i d e c h a i n combined w i t h a s i n g l e heme i s  c a l l e d a s u b u n i t o f hemoglobin.  I t i s e v i d e n t , however, t h a t t h e hemo-  g l o b i n s u b u n i t s and m y o g l o b i n have a s i m i l a r o v e r a l l s t r u c t u r e , and t h a t , more s p e c i f i c a l l y , t h e b a s i c l i n k a g e s between t h e heme and p r o t e i n a r e e s s e n t i a l l y t h e same (92-94).  I t i s hence f e l t t h a t hemoglobin i s  appropriately included i n this section. The p r o s t h e t i c group o f m y o g l o b i n and hemoglobins i s a protoheme ( F i g u r e 2.8) i n w h i c h t h e i r o n i s s t r o n g l y c o o r d i n a t e d t o t h e f o u r p y r r o l e n i t r o g e n s o f t h e p r o t o p o r p h y r i n IX. F e r r o - p r o t o p o r p h y r i n I X i s known as heme; F e r r i - p r o t o p o r p h y r i n IX~ as hemin'.  The f i f t h s i t e o f t h e heme i s l i n k e d t o  the i m i d a z o l e n i t r o g e n o f a h i s t i d i n e r e s i d u e , number 93 o f t h e m y o g l o b i n c h a i n , number 87 o f t h e hemoglobin a - c h a i n , and number 92 o f t h e hemoglobin 3-chain  (44,89,90).  The s i x t h , and l a s t , o f t h e i r o n c o o r d i n a t i o n s i t e s  i n heme i s a v a i l a b l e f o r oxygen o r l i g a n d b i n d i n g ( F i g u r e 2.9).  Directly  -35-  CH =CH  |  2  CHj  CHj  CH -CH -C00H 2  2  6 N  I Fe  3  X — N ,  5  F i g u r e 2.8. coordinate  Ferro-protoporphyrin  IX  (heme) and  its  system.  H i  1 — N —  H I l  0 ll II  c -C |  -  H — C -H i  1  C=C —  1 1 H —N  1 1  \ // C  N  HEME PLANE  s  Figure 2.9. Attachment of h i s t i d i n e F8. L r e p r e s e n t s position.  heme i r o n t o N « a l i g a n d i n the  of sixth  -36opposite to the iron-linked imidazole i s the " d i s t a l histidine", number 64 i n myoglobin, number 58 i n the hemoglobin a-chain, and number 63 i n the B-chain of hemoglobin.  I t has been suggested that the d i s t a l  histidine may play an important role i n the reversible oxygenation process. Recent crystallographic refinement of spermwhale metmyoglobin to o  o  2.OA shows that the iron atom i s displaced by 0.40A from the mean plane o  of the heme, Py, and the Fe-Ne (proximal histidine) i s 2.13A (95). The o  doming of the porphinato core i s 0.13A which i s i n agreement with that found i n 2-MeImFe(II)TPP (64). Takano (95) reported that the displacement o  of the iron atom from the mean plane of the heme increases from 0.40A to o  0.55A on going from met-to deoxy. The structure of oxy-Mb was recently solved.by P h i l l i p s i n 1978 (26).  The Fe atom out-of-plane displacement from the mean plane of o  the porphyrin i s 0.26A towards the proximal histidine (F8). Table 2.6 gives the heme geometry i n Mb and Hb derivatives. Recently G. Fermi (74) obtained a refined structure of human deoxyo  Hb at 2.5A resolution using the electron-density map obtained by Ten Eyck and Arnone (96). The displacement of the iron atom from Py i s less than that previously reported for horse deoxy-Hb (62), but the discrepancy  0 probably l i e s within the measurement errors.  The Fe...Py i s 0.60A i n the  o  o  a-heme and 0.63A i n the B-heme. The estimated error i s ±0.1A. With the o  available 2.5A data, Fermi could.not assess the doming magnitude accurately. Fermi, however, pointed out that the doming of the porphinate core i s o  unlikely to exceed the value of 0.17 to 0.22A. The Py...Ne (proximal o  o  histidine) i s 2.6A which i s compatible with the value of 2.68A quoted by Hoard and Scheidt (64) for high-spin ferrous five-corrdinate 2-MeImFe(II)TPP.  -37-  In horse met-Hb, B o l t o n and P e r u t z  (62) found t h e i r o n atom t o  be d i s p l a c e d from t h e mean plane o f t h e p o r p h y r i n  s k e l e t a l towards t h e  o  heme-linked h i s t i d i n e by 0.3A.  I n b o t h t h e a- and g-chains o f h o r s e o  deoxy-Hb t h i s displacement i s i n c r e a s e d  t o 0.75A.  formed p a r t o f t h e e x p e r i m e n t a l b a s i s f o r P e r u t z ' s of oxygen-binding c o - o p e r a t i v e The  phenomenon  This  observation  t r i g g e r mechanism  (6,29).  o v e r a l l shape o f myoglobin i s a f l a t t e n e d sphere.  The heme  group i s near t h e s u r f a c e o f the m o l e c u l e i n a n o n - p o l a r , hydrophobic pocket formed by t h e amino a c i d s , A l a , I l u , Phe., Leu, and V a l . 2.10  g i v e s t h e s t e r e o drawing o f myoglobin.  Figure  The presence o f s e v e r a l  n e a r l y a r o m a t i c r i n g s such as Phe p a r a l l e l t o t h e heme, i n t r o d u c e s t h e p o s s i b i l i t y o f ir-bonding i n t e r a c t i o n s .  The p l a n e o f t h e heme group i s  a p p r o x i m a t e l y normal t o t h e s u r f a c e of the myoglobin. van  d e r Waals c o n t a c t w i t h  The  h y d r o p h i l i c chains  the  solvent. The  83 atoms, o f t h e g l o b i n  The heme i s i n  (excluding  hydrogens).  a r e d i r e c t e d towards t h e s u r f a c e t o i n t e r a c t w i t h  s t r u c t u r e o f t h e n a t i v e p r o t e i n appears t o be h e l d  together  e s p e c i a l l y by non-polar i n t e r a c t i o n s between s i d e c h a i n s , and between s i d e c h a i n s and t h e heme group, w i t h p o l a r i n t e r a c t i o n s c o n t r i b u t i n g t o a l e s s e r extent.  About 70 t o 80 per cent  as an a - h e l i x . at very  of t h e p o l y p e p t i d e  In the a - h e l i c a l regions,  c h a i n i s arranged  t h e non-polar r e s i d u e s  r e g u l a r i n t e r v a l s , every 3.6 r e s i d u e s ,  repeat  so t h a t t h e s i d e o f t h e  h e l i x which f a c e s the i n t e r i o r o f t h e m o l e c u l e i s composed o f a row o f non-polar The  residues. X-ray s t u d i e s have r e v e a l e d  t h a t each o f t h e f o u r s u b u n i t s i n  hemoglobin has a s t r u c t u r e s i m i l a r t o t h a t of myoglobin, e s p e c i a l l y i n regard  t o the f o l d i n g o f the p o l y p e p t i d e  chain,  the number and d i s t r i b u t i o n  -38-  F i g u r e 2.10.  A three-dimensional  myoglobin. From D i c k e r s o n and G e i s  (Ref.97) •  stereo-pair  drawing  -39-  of t h e h e l i c a l and n o n - h e l i c a l segments, and t h e p o s i t i o n and o r i e n t a t i o n of t h e heme group. The f o u r hemoglobin s u b u n i t s a r e a r r a n g e d t o form a n e a r l y r e g u l a r tetrahedron  t o g i v e t h e whole m o l e c u l e a s p h e r i c a l appearance ( F i g u r e 2.11).  A g a i n t h e heme groups a r e near t h e s u r f a c e .  The a- and 3-chains a r e  complementary, and t h e s e c h a i n s a r e i n c l o s e c o n t a c t , i n c o n t r a s t t o the much l o o s e r a s s o c i a t i o n between t h e i d e n t i c a l  chains.  A major d i f f e r e n c e between m y o g l o b i n and hemoglobin i s t h a t m y o g l o b i n e x h i b i t s no c o - o p e r a t i v e  i n t e r a c t i o n w i t h molecular  oxygen.  -40-  Figure 2.11. hemoglobin.  The folding and packing of chains in  From Dickerson and Geis (Ref.97).  -41-  2.4  Models f o r M y o g l o b i n and Hemoglobin S e v e r a l groups o f i n v e s t i g a t o r s have developed models t h a t mimic  the a c t i v e - s i t e ( i . e . heme  o f m y o g l o b i n and hemoglobin. The s y n t h e t i c  complexes a r e c a p a b l e o f r e v e r s i b l e o x y g e n - b i n d i n g b u t i n non-aqueous media and o f t e n a t v e r y l o w t e m p e r a t u r e s , t y p i c a l l y -40°C. r e v i e w a r t i c l e on s y n t h e t i c o x y g e n - c a r r i e r s  i s by B. R. James (98) i n t h e  t r e a t i s e c o n s i s t i n g o f seven volumes on p o r p h y r i n s (99).  A good  e d i t e d by D.  Dolphin  S t u d i e s on such model systems would g i v e i n f o r m a t i o n about t h e  n a t u r e o f t h e m e t a l - d i o x y g e n bond and t h e a s s o c i a t e d k i n e t i c and thermodynamic f a c t o r s . I s o l a t e d heme i s r a p i d l y o x i d i s e d t o F e ( I I I ) w i t h t h e s i x t h of t h e i r o n c o o r d i n a t i o n s i t e s o c c u p i e d  site  by a h y d r o x y l i o n o r c e r t a i n  o t h e r l i g a n d s so t h a t i t cannot b i n d oxygen.~ The i r r e v e r s i b l e o x i d a t i o n t o F e ( I I I ) i s b e l i e v e d t o proceed through an i n t e r m e d i a t e i r o n porphyrins  i n w h i c h two  a r e c o v a l e n t l y l i n k e d known as y - o x o - b i s ( p o r p h y r i n ( I I I ) )  as shown i n F i g u r e 2.12. T h i s c r e a t e s a major o b s t a c l e i n d e s i g n i n g a s u i t a b l e h i g h - s p i n f i v e - c o o r d i n a t e F e ( I I ) complex as a p r o t e i n - f r e e model f o r m y o g l o b i n . y e a r s (100-102).  S e v e r a l models have been s y n t h e s i z e d  An important  i n the recent  p o i n t t o note i s t h a t a l l t h e model  compounds so f a r r e p o r t e d a r e monomers; hence, they a r e more c o r r e c t l y considered  t o be models o f m y o g l o b i n r a t h e r hemoglobin.  I n a l l of the  model systems, o x y g e n - b i n d i n g can be a c c o m p l i s h e d o n l y when t h e f i f t h s i t e o f t h e c o o r d i n a t i o n o f t h e m e t a l atom i s c o v a l e n t l y bound t o a base, g e n e r a l l y , a n i m i d a z o l e .  This leaves only the s i x t h coordination  s i t e o f t h e m e t a l atom a v a i l a b l e f o r o x y g e n - b i n d i n g .  -42-  Figure  2.12  A schematic drawing of y - o x o - b i s ( p o r p h y r i n i r o n  / \  N  N  \  /  0'  Fe  Fe F  N  I n h e m o g l o b i n and  m y o g l o b i n , t h e hemes a r e  h y d r o p h o b i c p o c k e t s as d i s c u s s e d studies  (III))  i n S e c t i o n 2.3.  ( 6 , 2 8 ) show t h e d i s t a n c e b e t w e e n any  two  N  embedded i n  non-polar,  X-ray c r y s t a l l o g r a p h i c i r o n atoms i n deoxy-Hb  o  o r oxy-Hb i s >25A; t h a t p r o b a b l y  e x p l a i n s why  t o F e ( I I ) atom i n h e m o p r o t e i n s w i t h o u t On  oxidizing  t h i s b a s i s , Collman's group at S t a n f o r d  with four bulky the r i n g l i k e porphyrin  o-pivalamidephenyl  stakes  c o n t a c t by  The  purpose of the bulky  t h e Fe a t o m s n e c e s s a r y  2.13  synthesized  for oxidation.  a porphyrin  "picket  Instead  ring  side  of  fence"  the " p i c k e t  F e ( I I ) complex w i t h  complexes undergo r e v e r s i b l e oxygenation slowly.  illustrates  binds  Fe(III).  s u b s t i t u e n t s i s to prevent  the a x i a l l i g a n d s i s produced.  o x i d a t i o n occurs  i r o n to  g r o u p s p r o j e c t i n g f r o m one  Figure  ordinate species, a hexa-coordinate b a s e s as  the  i n a p i c k e t f e n c e , h e n c e , known a s  (100,103,104,105).  porphyrin.  the oxygen molecule  fence"  the  of a f i v e  two  intimate co-  nitrogenous  I n b e n z e n e a t 20°C, t h e i m i d i a z o l e (98), although  irreversible  -43-  T r a y l o r ' s group (101) and B a l d w i n ' s group (102) have a l s o c o n s t r u c t e d s e v e r a l l y s t e r i c a l l y hindered  p o r p h y r i n s designed  These complexes a r e c a p a b l e o f r e v e r s i b l e  t o prevent d i m e r i z a t i o n .  oxygenation.  B a l d w i n and a s s o c i a t e s (102) r e p o r t e d t h e s y n t h e s i s of "capped" porphyrins  ( F i g u r e 2.14). The "cap" p r e v e n t s  c o o r d i n a t e s p e c i e s , and m o l e c u l a r protected s i t e .  t h e f o r m a t i o n o f hexa-  oxygen e n t e r s t h e c a v i t y on t h e  The complexes a r e c a p a b l e o f r e v e r s i b l e o x y g e n - b i n d i n g  i n p y r i d i n e a t 25°C. Chang and T r a y l o r (106) synthesized.an i n s i t u f e r r o u s complex such as shown i n F i g u r e 2.15 i n which an i m i d a z o l e i s c o v a l e n t l y bound to a p o r p h y r i n r i n g .  I t i s i n t e r e s t i n g t o n o t e t h a t t h e geometry o f  the complex i s s i m i l a r t o t h a t o f m y o g l o b i n . o c c u r s o n l y a t -45°C i n  dichloromethane.  R e v e r s i b l e oxygen-binding  -44-  F i g u r e 2.13.  "Picket-fence" porphyrin C o l l m a n e t a l . , (100,103-105)  F i g u r e 2.14.  "Capped" p o r p h y r i n s Baldwin e t a l . , (102)  —»-—«*»)„•  n  \ —C  CH--C  ^C-CNjCMjCOjt,  CH,  CH.  F i g u r e 2.15. C h e l a t e d hemes h a v i n g v a r y i n g d e g r e e s o f s t e r i c h i n d r a n c e toward base c h e l a t i o n . T r a y l o r e t a t . , (106).  -45CHAPTER I I I  EXPERIMENTAL WORK  3.1  P r e p a r a t i o n of a,g,Y,6-Tetraphenylphinatoindium(III) c h l o r i d e  Materials: Indium c h l o r i d e , I n C l ^ (anhydrous, u l t r a - p u r e ) was o b t a i n e d from A l f a ; m e s o - t e t r a p h e n y l p o r p h i n e was o b t a i n e d from Strem C h e m i c a l s , I n c . , and reagent g l a c i a l a c e t i c a c i d from A l l i e d Chemical Canada, L t d . S i l i c a g e l (70-140 mesh) f o r column chromatography  was o b t a i n e d from Macherey,  N a g e l & Co., Germany. Procedure: The procedure used t o s y n t h e s i z e t h e i n d i u m m e s o - t e t r a p h e n y l p o r p h y r i n c h l o r i d e ( I n T P P r C l ) was e s s e n t i a l l y t h a t o f B h a t t i e t a l . , o n e - l i t r e one-necked round-bottom  (107).  flask fitted with a reflux  In a  condenser,  i n d i u m c h l o r i d e (0.002M) and m e s o - t e t r a p h e n y l p o r p h i n e (0.001M) were heated to b o i l i n g i n g l a c i a l a c e t i c a c i d (500 ml) c o n t a i n i n g 2.2 gm o f sodium acetate.  The r e a c t i o n was p r o t e c t e d from l i g h t by aluminium  foil.  R e f l u x i n g was c o n t i n u e d f o r about 4 h r . C o m p l e t i o n o f t h e m e t a l a t i o n p r o c e s s was determined s p e c t r o p h o t o m e t r i c a l l y by t h e d i s a p p e a r a n c e o f the 515 nm peak c h a r a c t e r i s t i c o f t h e pure p o r p h i n e and t h e appearance of a peak i n t h e 560 nm r e g i o n c h a r a c t e r i s t i c o f t h e i n d i u m p o r p h i n e . Upon c o o l i n g t o room t e m p e r a t u r e , l u s t r o u s , p u r p l e c r y s t a l s o f I n T P P r C l were o b t a i n e d .  The p r o d u c t was c o l l e c t e d by vacuum f i l t r a t i o n , washed  w i t h methanol and a i r - d r i e d . The r e a c t i o n took p l a c e a c c o r d i n g t o e q u a t i o n 3.1 H (TPP) + I n C l 2  3  + 2Na(0Ac)  I n T P P r C l + 2H(0Ac) + 2NaCl  (3.1)  -46-  The o v e r a l l s t o i c h i o m e t r y i n Eq. 3.1,  looks rather simple.  In  f a c t t h e r e must be a t l e a s t f i v e s e p a r a t e p r o c e s s e s t a k i n g p l a c e i n Eq. 3.1.  (a)  They are as f o l l o w s :  Protonation/Deprotonation ?+  Equilibria  -4-?H~'~  _9ii"'~  Z^h  H.(TPP) 4  H„(TPP) 7  o_  >  (TPP)  (3.2)  t  £  2D e p r o t o n a t i o n of H (TPP) w i l l g i v e a d i a n i o n , ( T P P )  , which w i l l  2  r e a d i l y complex w i t h a p o s i t i v e l y charged metal porphine was was  first  obtained.  dissolved  From Eq. 3.2,  As the r e a c t i o n  l a t i o n by s h i f t i n g the e q u i l i b r i u m to the l e f t .  At f i r s t  progressed,  o b t a i n e d i n d i c a t i n g the f o r m a t i o n of  i t i s clear that strong acids w i l l  a c e t i c a c i d was  meso-tetraphenyl-  a t t r i b u t e d to the f o r m a t i o n of a  (See Appendix B ) .  a deep p u r p l e s o l u t i o n was  When  i n g l a c i a l acetic acid, a greenish s o l u t i o n  The g r e e n i s h c o l o r was 2+  d i a c i d s p e c i e s , H^(TPP)  ion.  impede or r e v e r s e m e t a l A weak a c i d such as  glacial  used. g l a n c e , Eq. 3.2  suggests  t h a t b a s i c s o l v e n t s s h o u l d be  more a p p r o p r i a t e s o l v e n t s than a c i d s because they w i l l d r i v e the t o n a t i o n e q u i l i b r i u m to the r i g h t . behave as Lewis bases which may donors to the metal  the l e f t ,  InTPP:Cl.  ions.  thus impeding  Sodium a c e t a t e was  the  depro-  However, the b a s i c s o l v e n t molecules  more or l e s s s t r o n g l y b i n d as n e u t r a l  This w i l l  shift  the e q u i l i b r i a i n Eq. 3.3 3+  the f o r m a t i o n of "naked" In added to b u f f e r the s o l u t i o n and  d e p r o t o n a t i o n of the p o r p h y r i n s by s h i f t i n g  to  metal i o n s . to enhance  the e q u i l i b r i u m  (Eq. 3.2)  to  the r i g h t . I(b) n C l - D+ In(OAc)., e c o3Na(0Ac) n v o l u t i o n of the metal +i o3NaCl n from the m e t a l s  carrier  (3.3a)  -47-  In(OAc)  The In  3+  3  ,  IrT  k  formation  r r  + 3(0Ac)  of I n ( T P P )  (3.3b)  depends on the a v a i l a b i l i t y  +  i o n s t h a t w i l l complex w i t h  " b a r e " (TPP)  2-  .  of "naked"  This  implies  3+ t h a t i n d i u m a c e t a t e has may  to d i s s o c i a t e r e a d i l y to give In  ions.  This  be a v e r y d e c i s i v e s t e p i n the m e t a l a t i o n r e a c t i o n . (c) F o r m a t i o n of t h e e q u a t o r i a l MN^ species In  +  3 +  ( T P P ) ^ = ^ In(TPP) 2 _  (3.4)  +  Complexation between a " b a r e " In~ ~ i o n and rT  the d i a n i o n  (TPP)^  is  a straightforward reaction. (d)  N e u t r a l i z a t i o n of the charge  In(TPP)  +  + Cl"  To m a i n t a i n  * InTPPrCl  (3.5)  e l e c t r o n e u t r a l i t y , an a n i o n i s bound t o the m e t a l i o n .  I t i s a s t o n i s h i n g t h a t i n s p i t e of the l a r g e excess of a c e t a t e a c e t a t e complex was (e)  not  present,  formed.  C o m p l e t i o n of the c o - o r d i n a t i o n  sphere  3+ The  In  i s c o o r d i n a t e l y s a t u r a t e d , thus o n l y f i v e - c o o r d i n a t e square-  pyramidal InTPPrCl coordinate  i s formed.  I n some m e t a l complexes, s i x - , and  s p e c i e s are p o s s i b l e .  eight-  -48-  3.2a  P u r i f i c a t i o n of  InTPP:Cl  The p u r i f i c a t i o n p r o c e d u r e f o r I n T P P r C l was of B h a t t i e t a l . ; (107). minimal  The crude complex (100 mg)  toluene/chloroform/chloroform  f i r s t , f o l l o w e d by I n T P P r C l , and by t i c . recover 3.2b  The column was  m i x t u r e , the u n r e a c t e d  dissolved i n cm  eluted with a TPP was  eluted  A l l f r a c t i o n s were checked s p e c t r o s c o p i c a l l y  The s o l v e n t was  evaporated  on a vacuum r o t a r y e v a p o r a t o r  to  InTPPrCl.  T h i n - l a y e r Chromatography T h i n - l a y e r chromatography was  p u r i f i e d InTPPrCl.  used to develop  used t o check the p u r i t y of the  A n a l y t i c a l t h i n p l a t e s of s i l i c a g e l (Eastman) were  s p o t t e d w i t h 5 y l samples.  TLC  was  from t h a t  amount of c h l o r o f o r m and chromatographed on a 62 cm x 1.6  column o f s i l i c a g e l packed i n t o l u e n e . 1:1  different  A m i x t u r e of t o l u e n e / c h l o r o f o r m  (1:1)  was  the chromatogram. The chromatograms were r u n i n t h e  dark.  showed the absence of t h e s t a r t i n g m a t e r i a l , meso-TPP i n t h e p u r i f i e d  InTPPrCl. 3.2c  V i s i b l e Absorption  Spectroscopy  The v i s i b l e a b s o r p t i o n s p e c t r a of I n T P P r C l and meso-TPP i n c h l o r o f o r m were r e c o r d e d on a Cary 14 s p e c t r o m e t e r  ( F i g u r e 3.1  and F i g u r e 3.2).  The  meso-TPP shows f o u r a b s o r p t i o n bands ( e x c l u d i n g the S o r e t band) i n t h e v i s i b l e r e g i o n , l a b e l l e d I-IV ( F i g u r e 3.2). (e>10  5  ) c o u l d o n l y be determined  The v e r y i n t e n s e S o r e t band  a t a c o n c e n t r a t i o n of VLO  —6  M.  On f o r m a t i o n of I n T P P r C l , t h e four-banded spectrum c o l l a p s e d i n t o an e s s e n t i a l l y two-banded one  (a and 8 bands) i n the v i s i b l e r e g i o n ,  whereas t h e S o r e t band remained.  These bands a r e b a t h o c h r o m i c a l l y  s h i f t e d , the a-band o c c u r r i n g a t 600 nm, S o r e t band a t 419 nm;  the 8-band a t 560 nm,  the i n t e n s i t y sequence i s S o r e t >>g>a.  and  the  -49-  426  1  L  384  460  J  306  I  474  Wavelength F i g u r e 3.1. chloroform.  V i s i b l e absorption  I  i  j  549  624  700  nm spectrum of  InTPP:Cl  in  -50-  i IV  515  310  F i g u r e 3.2. chloroform.  384 460 471 Wavelength nm  V i s i b l e absorption  547  spectrum of  639  700  meso-TPP  in  -51-  3.2d  N u c l e a r Magnetic  Resonance  Spectroscopy  The n.m.r. spectrum o f I n T P P r C l i n d e u t e r o c h l o r o f o r m was r e c o r d e d on a V a r i a n XL-100 FT-NMR s p e c t r o m e t e r , w i t h 3.3  2 D-Lock on CDC1 . 3  R e c r y s t a l l i s a t i o n of InTPPrCl S p e c t r o q u a l i t y c h l o r o f o r m was o b t a i n e d from MCB, and s p e c t r o -  methanol was o b t a i n e d from Eastman. R e c r y s t a l l i z a t i o n was c a r r i e d out by d i s s o l v i n g t h e m e t a l l o p o r p h y r i n i n a m i n i m a l volume o f c h l o r o f o r m , and adding methanol dropwise boiling solution.  to the  W i t h c o n t i n u e d b o i l i n g , c h l o r o f o r m d i s t i l l e d away,  and t h e methanol c o n c e n t r a t i o n i n c r e a s e d ; t h e p r o c e s s was a l l o w e d t o c o n t i n u e u n t i l some s m a l l c r y s t a l s formed.  Purple  single  crystals  were o b t a i n e d by l e t t i n g t h e m i x t u r e s t a n d f o r s e v e r a l days a t room temperature.  -52-  CHAPTER IV RESULTS AND  4.1  Metal displacement The  DISCUSSION  above mean p y r r o l e n i t r o g e n p l a n e  c r y s t a l s are shown t o be m o n o c l i n i c w i t h a space group of P2^/n.  The numbering scheme used f o r the carbon and n i t r o g e n atoms i n the asymmetric u n i t of c r y s t a l l i n e I n T P P r C l i s shown i n F i g u r e 4.1. u n i t c e l l a r e shown i n F i g u r e The  stereo-drawing  displacement  The  c o n t e n t s of  the  4.2.  of InTPP:Cl  i n F i g u r e 4.3  c l e a r l y shows the  of the indium atom above the mean plane of the f o u r p y r r o l e  n i t r o g e n atoms, and a l s o s i g n i f i c a n t doming of the p o r p h i n a t o  core.  The  o  indium atom o u t - o f - p l a n e d i s p l a c e m e n t  i s 0.71A  from t h e mean p l a n e of the  o  p o r p h y r i n w i t h 0.1A  a t t r i b u t e d to the "doming".  I n T P P r C l i s not  planar  but e x h i b i t s a " r u f f l i n g " of the p o r p h y r i n s k e l e t o n . A number of i n t e r e s t i n g f e a t u r e s emerge from t h e s t e r e o c h e m i c a l of v a r i o u s m e t a l l o p o r p h y r i n s l i s t e d i n o r d e r of d e c r e a s i n g displacements  (Table 4.7)r  data  metal  (a) t h e r e i s so s i m p l e c o r r e l a t i o n between the  i o n i c r a d i u s and m e t a l i o n d i s p l a c e m e n t ;  (b) the d i s p l a c e m e n t s  f o r the same  m e t a l c o o r d i n a t e d t o d i f f e r e n t p o r p h y r i n s a r e s i m i l a r as e v i d e n t from  Co-  o  atom d i s p l a c e m e n t and  the  of 0.13A  both i n l-MeImCo(II)TPP (54) and 1-MeImCo(II)OEP ( 5 5 ) ,  v a r i o u s h i g h - s p i n f e r r i c p o r p h y r i n complexes a l s o have s i m i l a r  Fe...Py d i s t a n c e s ; and  (c) i n d i u m ( I I I ) atom i s d i s p l a c e d e s s e n t i a l l y  same d i s t a n c e as i n h i g h - s p i n F e ( I I ) i n deoxy-Hb (horse or human). inferred  the I t i s often  t h a t h i g h - s p i n s t a t e s i m p l y f i v e - c o o r d i n a t i o n , w h i c h so f a r i s  v a l i d f o r f e r r o u s p o r p h y r i n s and hemoproteins.  I n 1978, Mashiko et a l . ,  ( I l l ) r e p o r t e d the f i r s t s t r u c t u r a l c h a r a c t e r i s a t i o n o f s i x - c o o r d i n a t e h i g h - s p i n f e r r i c p o r p h y r i n d e r i v a t i v e s of TPP  i n which the F e ( I I I ) atom i s  Figure  4.1.  Atomic numbering system f o r c r y s t a l l i n e I n T P P r C l .  atoms a r e drawn as 50% p r o b a b i l i t y t h e r m a l h a v e been o m i t t e d f o r  c l a r i t y . The p h e n y l  w h e r e n i s t h e g r o u p number  (n=l , 4 )  ellipsoids.  The  The H atoms  r i n g s a r e numbered a s  and m i s t h e r i n g c a r b o n  nCm>  (m=l,6).  Figure 4 . 2 . cell  of  S t e r e o s c o p i c view of  crystalline  InTPP:C1.  the contents of  one  unit  F i g u r e 4.3.  Stereo  displacement  from the m a c r o c y c l i c p l a n e ,  the  porphyrin  reduced f o r  diagram of  skeleton. clarity.  The  InTPPrCl,  H atoms  i l l u s t r a t i n g the  In  and t h e  of  have been  "doming"  arbitrarily  -56-  p r e c i s e l y i n t h e mean p o r p h y r i n p l a n e .  S i x - c o o r d i n a t i o n i s a l s o known  i n h i g h - s p i n f e r r i c hemoproteins as i l l u s t r a t e d by aquo- and f l u o r o methemoglobin.  I n c o n t r a s t , t h e F e ( I I I ) atoms a r e s i x - c o o r d i n a t e and  l o w - s p i n i n CN-MetHb and CO-MetHb. The  observation  (b) emphasises t h e f a c t t h a t t h e m e t a l d i s p l a c e m e n t  i s s i m i l a r i n a l l metalloporphyrin t o assume w i t h c o n f i d e n c e  behavior  complexes o f a s i n g l e m e t a l type. I t i s l o g i c a l  t h a t subsequent f i v e - c o o r d i n a t e indium  porphyrin  complexes w i l l e x h i b i t s i m i l a r d i s p l a c e m e n t o f indium atoms above t h e mean p o r p h y r i n Perutz  plane.  (28,29) proposed a s t e r e o c h e m i c a l  co-operative nature  of oxygen-binding.  mechanism t o e x p l a i n t h e  The a l l o s t e r i c model was based on  the d i f f e r e n c e s i n t h e t e r t i a r y and q u a t e r n a r y and met-Hb, r a t h e r than deoxy- and oxy-Hb.  s t r u c t u r e s o f horse deoxy-  Oxy-Hb i s r e a d i l y o x i d i s e d t o  met-Hb under t h e normal c o n d i t i o n s o f X-ray c r y s t a l l o g r a p h y .  Perutz  j u s t i f i e d h i s argument on t h e ground t h a t oxy-Hb and met-Hb a r e isomorphous i . e . t h e c r y s t a l s have t h e same space group.  H u e s t i c and R a f t e r y (112)  19 showed t h a t t h e chemical CO-Hb.  F - l a b e l l e d c y s t e i n e 938 i n met-Hb g i v e s a n.m.r.  s h i f t s i m i l a r t o deoxy-Hb b u t was d i f f e r e n t from oxy- and T h i s i m p l i e s t h a t met-Hb i s not a good analog f o r oxy-Hb.  Besides  the F e ( I I I ) i s s i x - c o o r d i n a t e and p r e d o m i n a n t l y h i g h - s p i n i n met-Hb as opposed t o F e ( I I ) which i s l o w - s p i n and s i x - c o o r d i n a t e i n oxy-Hb. P e r u t z i n 1976 (113) proposed t h a t CO-Hb "may be regarded as t h e c l o s e s t r e l a t i v e of oxy-hemoglobin" on the ground t h a t CO-Hb i s s t a b l e and isomorphous w i t h b o t h oxy- and met-Hb.  F u r t h e r m o r e , t h e F e ( I I ) atoms i n oxy- and CO-Hb o  are l o w - s p i n . However, t h e 2.8A X-ray data on CO-Hb i s not s u f f i c i e n t t o d e t e r m i n e the exact d i s p l a c e m e n t o f t h e i r o n atoms from t h e heme p l a n e . The  s t r u c t u r e o f spermwhale oxymyoglobin has been determined by P h i l l i p s i n  -.57-  1978  (26).  P h i l l i p s c l e a r l y demonstrates t h a t t h e Fe atom does move  towards t h e p o r p h y r i n on t r a n s i t i o n from deoxy-to oxy-Mb (See S e c t i o n 2.2 and T a b l e 2.6).  This finding  r e i n f o r c e s P e r u t z ' s a l l o s t e r i c model  a l t h o u g h t h e t o t a l movement o f the p r o x i m a l h i s t i d i n e i n Mb i s s m a l l e r than t h a t expected Perutz  f o r Hb.  (28,29) proposed t h a t t h e q u a t e r n a r y  i s i n e q u i l i b r i u m between "T"  s t r u c t u r e o f hemoglobin  two s t a b l e s u b u n i t c o n f i g u r a t i o n s termed as  f o r deoxy-Hb and "R" f o r oxy-Hb.  I n t h e deoxy-Hb, t h e l o w - s p i n , o  f i v e - c o o r d i n a t e heme i r o n atom i s ^0.75A (horse hemoglobin) out o f t h e heme p l a n e towards t h e p r o x i m a l h i s t i d i n e .  When a m o l e c u l e  o f oxygen  b i n d s t o deoxy-Hb, t h e i r o n moves i n t o t h e p l a n e o f t h e p o r p h y r i n . A c c o r d i n g t o P e r u t z ' s s t e r e o c h e m i c a l mechanism, t h e t r a n s i t i o n from "T"  s t a t e (deoxy-Hb) t o "R" s t a t e (oxy-Hb) i n v o l v e s a s h r i n k a g e o f  M3.90A i n t h e Pu...Ne d i s t a n c e . s m a l l changes i n t h e q u a t e r n a r y  T h i s movement t r i g g e r s a s e r i e s o f and t e r t i a r y s t r u c t u r e s of t h e p r o t e i n  that r e s p o n s i b l e f o r t h e oxygen-binding  co-operativity.  At the present  t h e r e i s s t i l l a c o n s i d e r a b l e debate and c o n t r o v e r s y s u r r o u n d i n g t h e q u e s t i o n o f t h e s t e r e o c h e m i c a l mechanism. Perutz advocates t h a t  There a r e two s c h o o l s o f thought:  t h e changes i n t h e s t e r e o c h e m i s t r y a t t h e  c o r e o f the heme a r e t h e key t o t h e t r i g g e r mechanism w h i l e t h e o t h e r s c h o o l ( E d e l s t e i n and Gibson importance ligand.  ( 8 1 ( b ) ) , and L i t t l e and I b e r s (55))emphasises  the  o f t h e i n t e r a c t i o n between t h e d i s t a l h i s t i d i n e and t h e s i x t h -  A c r i t i c a l t e s t f o r t h e t r i g g e r mechanism would be t o " f r e e z e "  one o r more s u b u n i t s i n t o t h e "T" s t a t e and then m o n i t o r oxygenation  changes i n t h e  k i n e t i c s , e l e c t r o n i c , and m o t i o n a l p r o p e r t i e s o f t h e r e m a i n i n g  i n t a c t n a t i v e s u b u n i t o r s u b u n i t s . N a t i v e s u b u n i t s i n t h e "T" s t a t e a r e v e r y  -58d i f f i c u l t t o a c h i e v e s i n c e heme F e ( I I ) b i n d s oxygen t o become hexa-. c o o r d i n a t e and l o w - s p i n t o g i v e t h e "R" s t a t e . S i n c e t h e "T" form cannot be s t a b l y generated u s i n g n a t i v e i r o n heme ( r e a d i l y a u t o x i d i s e s t o F e ( I I I ) ) , o t h e r means must be sought. V a r i o u s m e t a l l o p o r p h y r i n s c o n t a i n i n g Mn, Co, Zn, and Mg i n p l a c e o f n a t i v e heme have been r e c o n s t i t u t e d i n t o hemoglobin.  However, M n ( I I )  i s r e a d i l y o x i d i s e d t o M n ( I I I ) , and C o ( I I ) b i n d s oxygen.  Both Mg and  Zn may be o x i d a t i o n s t a b l e b u t they e x h i b i t r e l a t i v e l y s m a l l m e t a l displacements  above t h e p o r p h y r i n p l a n e r e l a t i v e t o t h e Fe atom i n n a t i v e  hemoglobin (See T a b l e 4.7).  The p r e s e n t X-ray d a t a on InTPP:Cl  suggest  indium p o r p h y r i n s a r e i d e a l c a n d i d a t e s f o r i n d u c i n g t h e "T" c o n f o r m a t i o n (vive i n f r a ) .  I n c o r p o r a t i o n o f indium p o r p h y r i n s i n t o hemoglobin p r o v i d e  an o p p o r t u n i t y t o probe the p r o p h y r i n - a p o p r o t e i n i n t e r a c t i o n i n t h e "T" s t a t e hemoglobin and myoglobin. Hoffman and a s s o c i a t e s (83-86) have shown t h a t c o b a l t - s u b s t i t u t e d m y o g l o b i n and hemoglobin can combine r e v e r s i b l y and c o - o p e r a t i v e l y w i t h m o l e c u l a r oxygen, a l t h o u g h , t o a much l e s s e r e x t e n t than t h e n a t i v e proteins.  I t i s i n t e r e s t i n g t o note i n T a b l e 4.7 t h a t a l t h o u g h  Co(II)  o  has a l a r g e r i o n i c radium (0.72A) than F e ( I I I ) , the o u t - o f - p l a n e  dis-  placement o f Co atom from t h e mean plane o f t h e p y r r o l e n i t r o g e n s i s o n l y o  0.13A.  o  I n c o n t r a s t , t h e s m a l l e r F e ( I I I ) ( i o n i c r a d i u s = 0.64A) has a o  l a r g e r metal displacement  o  r a n g i n g from 0.38A t o 0.52A.  s t u d i e s on CoHb and Hb by Woodruff et a l . ,  Resonance Raman  (87) i n d i c a t e t h a t t h e C o ( I I ) o  atom o u t - o f - p l a n e d i s p l a c e m e n t  i n Co-Hb cannot exceed 0.2A.  model by P e r u t z on t h e mechanism o f r e v e r s i b l e o x y g e n - b i n d i n g i n hemoglobin depends d i r e c t l y on t h e l a r g e change on the upon o x y g e n a t i o n  (See S e c t i o n 2.2).  The a l l o s t e r i c co-operativity  Pp...Ne  distance  I t i s thus l o g i c a l t o wonder whether  -59-  t h e P e r u t z ' s " t r i g g e r " mechanism i s c o n s i s t e n t w i t h the much s m a l l e r out-of-plane displacement  of C o ( I I ) i n model m e t a l l o p o r p h y r i n s  and  CoHb. The most r e a s o n a b l e  r a t i o n a l i s a t i o n of t h i s dilemma i s t h a t of  Hoard and  propose t h a t the t e n s i o n w i t h i n the p r o t e i n  Scheldt  (64) who  i n deoxy-CoHb causes the p o r p h i n a t o  c o r e to undergo a s u b s t a n t i a l  o  doming of MJ.5A towards the Co atom.  The  doming r a i s e s the Co atom  o  out-of-plane displacement  o  t o ^0.70A c o m p a t i b l e  t o Fe atom (0.75A) i n  o  h o r s e deoxy-Hb and and  0.6A  Scheidt proposal  i n human deoxy-Hb.  A support  f o r the Hoard  (64) comes from the f a c t t h a t t h e  s k e l e t o n i s r e a d i l y deformed normal t o the mean p l a n e .  prophyrin As one  example,  Ni(OEP) c r y s t a l l i s e s i n b o t h a t r i n c l i n c i c form and a t e t r a g o n a l form (35).  Both c r y s t a l s a r e v i r t u a l l y isomorphous, but y e t the t e t r a g o n a l  form e x h i b i t s marked n o n - p l a n a r i t y of the p o r p h i n a t o triclinic achieve  form i s e s s e n t i a - l y p l a n a r .  the " t e n s e " q u a t e r n a r y  between the C o ( I I ) atom and  core w h i l e  the  CoHb i s t h e r e f o r e thought t o  s t r u c t u r e , not so much by the  separation  the mean p o r p h y r i n p l a n e , but r a t h e r by  marked "doming" o f the p o r p h y r i n s k e l e t o n i t s e l f t o move the c o b a l t nearer  t o the heme-linked h i s t i d i n e The  "R"  oxygen-binding, expected  s t a t e quarternary  (F8).  s t r u c t u r e s of CoHb and FeHb ( i . e . ,  after  so t h a t the m e t a l atom l i e s i n the p o r p h y r i n p l a n e ) a r e  t o be s i m i l a r , w h i l e the o v e r a l l f u n c t i o n a l p r o p e r t i e s a r e v e r y  different.  For example the o x y g e n - a f f i r i t y o f Co-Hb i s reduced by 10-100  t i m e s r e l a t i v e t o n a t i v e Hb and Mb  (86).  F u r t h e r m o r e , Collman  and  a s s o c i a t e s (124) have shown t h a t the f r e e energy d i f f e r e n c e between the i n t r i n s i c b i n d i n g of t h e f i r s t o f Hb.  Therefore,  and  f o u r t h oxygen t o CoHb i s o n e - t h i r d t h a t  i t seems r e a s o n a b l e  to conclude  t h a t the "T"  s t a t e quarter-  nary s t r u c t u r e s of CoHb and Hb must be a p p r e c i a b l y d i - f e r e n t , most because o f the much s m a l l e r d i s p l a c e m e n t  of Co atom above the  probably  porphyrin  -60-  plane.  I n seeking a metalloporphyrin  apo-hemoglobin, w i l l produce a " t e n s e "  w h i c h , when r e c o n s t i t u t e d i n t o c o n f o r m a t i o n more s i m i l a r t o  t h a t f o r t h e n a t i v e i r o n - c o n t a i n i n g p r o t e i n , one t h e r e f o r e seeks a case f o r w h i c h t h e m e t a l d i s p l a c e m e n t above the p o r p h y r i n p l a n e i s more s i m i l a r t o t h a t f o r t h e c o r r e s p o n d i n g h i g h - s p i n F e ( I I ) complex. From T a b l e 4.7, i t i s c l e a r t h a t t h e r e a r e r e l a t i v e l y few a t t r a c t i v e candidates. al.,  Mn-Hb has been p r e p a r e d ( 8 0 ) , and M o f f a t e t  (79) c o n c l u d e d from t h e i r c r y s t a l l o g r a p h i c s t u d i e s on Mn-Hb t h a t  deoxy-Mn(II)Hb w i l l adopt t h e normal "T" q u a t e r n a r y s t r u c t u r e . However, Mn(II)-Hb i s i r r e v e r s i b l y o x i d i s e d t o M n ( I I I ) - H b which has been shown t o be a c l o s e s t r u c t u r a l a n a l o g o f met-Hb ( 7 9 ) . Mg and Zn (76), w h i l e presumably s t a b l e toward o x i d a t i o n , e x h i b i t r e l a t i v e l y s m a l l m e t a l d i s p l a c e m e n t s above t h e p o r p h y r i n  p l a n e , and may t h u s n o t be a p p r e c i a b l y  b e t t e r than Co i n t h i s r e s p e c t . The l a r g e i o n s , T l , H f , Z r , and B i , g i v e d i s p l a c e m e n t s which may be t o o l a r g e (compared t o F e ) , and i n t h e cases of Hf and Z r g i v e u n s u i t a b l e c o o r d i n a t i o n number.  However, t h e p r e s e n t  r e s u l t s suggest t h a t an indium p o r p h y r i n may be an i d e a l c a n d i d a t e f o r inducing the "tense"  c o n f o r m a t i o n i n hemoglobin, w i t h o u t t h e c o m p l i c a t i o n  of a u t o x i d a t i o n , o x y g e n - b i n d i n g t o t h e i n d i u m - l a b e l l e d s u b u n i t s , and w i t h the n e a r - i d e a l d i s p l a c e m e n t o f the m e t a l atom from t h e mean  porphyrin  plane. Indium has a f u r t h e r advantage t h a t i t has an i s o t o p e i s s u i t a b l e f o r gamma-gamma c o i n c i d e n c e measurements. thesis describes  the r e c o n s t i t u t i o n of  ("''"'""'"In) w h i c h  P a r t two o f t h i s  InMPP I X i n t o apomyoglobin,  and how gamma-gamma a n g u l a r c o r r e l a t i o n measurements can g i v e p r o p e r t i e s of the p r o t e i n .  motional  -61-  4.2  Bond Lengths  and  Angles  The bond l e n g t h s and a n g l e s of T a b l e 4.5  show t h a t t h e  p y r a m i d a l InTPP:Cl m o l e c u l e has v e r y c l o s e t o C ^  square  symmetry, as f a r as the  I n , C l , and p o r p h y r i n s k e l e t o n atoms a r e concerned.  (The p h e n y l  tilts o  a r e d i s c u s s e d i n S e c t i o n 4.4).  The average In-N d i s t a n c e i s 2.156(6)A,  which i s s u b s t a n t i a l l y l a r g e r than f o r the c o r r e s p o n d i n g h i g h - s p i n F e ( I I I ) o  complexes (Ca. 2.06-2.09A).  T h i s i n c r e a s e i s due p a r t l y t o expansion  of  o  the p o r p h y r i n c o r e ( r a d i u s of 2.067A f o r I n T P P C l , compared t o a p p r o x i m a t e l y 2.00  t o 2.04  f o r i r o n ( I I I ) ) , and p a r t l y t o an i n t r i n s i c a l l y l o n g e r  bond compared t o Fe-N bond.  However, the Fe-N bond l e n g t h f o r i r o n i n  hemoglobin i s expected t o i n c r e a s e t o a p p r o x i m a t e l y the p r e s e n t v a l u e , due t o t e n s i o n produced (64,65,55);  In-N  In-N  by t h e p r o x i m a l h i s t i d i n e l i g a n d of Hb  thus the indium p o r p h y r i n may  s t i l l be a c l o s e s t r u c t u r a l  a n a l o g f o r F e ( I I ) i n deoxyhemoglobin, even though the i s o l a t e d p o r p h y r i n s t r u c t u r e s appear r a t h e r d i f f e r e n t w i t h r e s p e c t to m e t a l - n i t r o g e n bond lengths.  -62-  4.3  Doming of the P o r p h y r i n  Skeleton o  The M-Np for  minimal  d i s t a n c e of 2.01A  appears t o be a n e a r l y optimum v a l u e  s t r a i n and u n d i s t o r t e d accomodation of the m e t a l atom  w i t h i n the c e n t r a l " h o l e " of the p o r p h y r i n .  Structural studies  have shown t h a t f o r a h i g h l y expanded p o r p h i n a t o non-planar  conformation  c o r e , the most e f f i c i e n t  i s doming of the c o r e (18,36).  (15) a l s o have demonstrated e x p e r i m e n t a l l y  (21)  Hoard and  the h i g h degree of  o f the p o r p h y r i n s k e l e t o n i n m e t a l l o p o r p h y r i n s t r u c t u r e s .  The  coworkers  flexibility long  o  (In-Np)  a v  d i s t a n c e , 2.156A w h i c h i s undoubtedly r e s p o n s i b l e f o r the  s i g n i f i c a n t "doming" of the p o r p h i n a t o core i s c l e a r l y i l l u s t r a t e d i n the s t e r e o - d r a w i n g InTPP:Cl  i n F i g u r e 4.3.  i s n o n - p l a n a r , and  of the p o r p h y r i n atoms.  I t i s e v i d e n t from F i g u r e 4.3  e x h i b i t s a marked d e v i a t i o n s from p l a n a r i t y  Atomic d i s p l a c e m e n t s  from the mean p o r p h y r i n  p l a n e o f InTPP:Cl a r e i l l u s t r a t e d i n F i g u r e 4.4. displacements  that  The  ( p o s i t i v e and n e g a t i v e s i g n correspond  perpendicular to  displacement  above ( i . e . , toward the indium) or below the plane) from the mean p l a n e o f a l l 28 core p o r p h y r i n atoms i n F i g u r e 4.4  show t h a t the p o r p h y r i n  s k e l e t o n i s a p p r e c i a b l y "domed" upward toward the indium.  The  plane of o  the f o u r p y r r o l e n i t r o g e n atoms i s i n f a c t d i s p l a c e d an average of 0.1A the mean p l a n e of the c o r e of the p o r p h y r i n atoms.  above  The outermost p y r r o l e  o  r i n g carbons a r e depressed  by up to 0.214A below the  plane. o  I t i s i n t e r e s t i n g t o note t h a t the doming i n 2-MeImFe(II)TPP i s  0.13A  o  w h i l e i t i s 0.1A  f o r InTPPrCl.  T h i s suggests  t h a t the p o r p h y r i n s k e l e t o n  i s not s i g n i f i c a n t l y d i s t o r t e d by the presence of indium atom.  Fermi  (74)  p o i n t e d out t h a t the doming of the p o r p h i n a t o core i n human deoxy-Hb i s o  u n l i k e l y t o exceed the v a l u e of 0.17  to 0.22A.  I n T P P r C l i s w e l l w i t h i n the range observed  The  degree of doming i n  for high-spin f e r r i c  porphyrins  -63-  (Table 4.7).  I t i s expected the i n f l u e n c e by the p r o t e i n environment  i n c r e a s e the doming of indium p o r p h i n a t o c o r e c o m p a t i b l e t o t h a t i n native  hemoglobin.  may  _  Figure  4.4.  porphyrin  Atomic  plane  82  ——5  51  displacements ( A x  (plane  #2 o f  Table  4.6)  10 of  ) f r o m t h e mean InTPP:Cl.  numbers c o r r e s p o n d t o d i s p l a c e m e n t s t o w a r d t h e  indium  Positive atom.  -65-  4.4  Phenyl Rings: C r y s t a l Packing A stereo view of a u n i t c e l l i s shown i n Figure 4.2, and  the moelcular packing.  illustrates  The c l o s e s t nonbonded intermolecular contact  o  i s 2.48A between H4C4 and H3C3 at (1-x, 1-y, z ) , i n d i c a t i n g that there i s no strong hydrogen bonding network and only the normal van der Waals forces between molecules. I t i s of i n t e r e s t to note that phenyl r i n g #1 i s close to an i n v e r s i o n center, and as a r e s u l t i t i s twisted by only 66.17° with respect to the mean plane of the porphyrin skeleton, i n order to minmize the nonbonded contacts to i t s centrosymmetrically r e l a t e d image.  The other three rings  have d i h e d r a l angles with respect to the porphyrin mean plane which are c l o s e r to perpendicular (107.59°, 93.67°, and 98.33° for phenyl rings Z/2-//4, r e s p e c t i v e l y ) . That the four phenyl r i n g s are equivalent i n s o l u t i o n i s shown by the proton NMR  spectrum of InTPPCl i n Figure 4.5.  When t h i s spectrum was  f i r s t observed (107), the magnetically inequivalent ortho protons of the phenyl r i n g s were i n c o r r e c t l y ascribed to "non-equivalence of phenyls i n p a i r s " , presumably due to two chemically d i f f e r e n t sets of two phenyls each. The present c r y s t a l s t r u c t u r e i n d i c a t e that the inequivalence a r i s e s from the displacement  of the indium above the porphyrin r i n g , making the upper  (four) and lower (four) ortho protons inequivalent.  The coalescence of  these two ortho proton peaks on increase i n temperature (125,126) must thus a r i s e from i n t e r n a l r o t a t i o n of the chemically equivalent phenyl groups about the bonds which connect the phenyl rings to the porphyrin.  -66-  InTPPCI  d e b 1  *  1  10 Figure 4 . 5 . solvent. (b), (8.20  ]  H  100 MHz FT-NMR s p e c t r u m o f I n T P P : C l i n C D C 1 3  Peak a s s i g n m e n t s  m- a n d p - p h e n y l and 8 . 4 3 ) ;  are: (a),  protons  (7.87);  and ( e ) , p y r r o l e  obtained using a Varian  residual  XL-100  C H C 1 3 (6 = 7 . 3 3 ) ;  ( c ) and ( d ) ,  g-protons  o-phenyl  (9.15).  FT-NMR s p e c t r o m e t e r ,  S p e c t r u m was with  2 used f o r  D lock,  sensitivity  10 t r a n s i e n t s ,  enhancement  6 . 0 sec pulse delay,  time constant  of - l . o sec.  protons  and  CDCU  -67-  Table 4 . 1 .  C r y s t a l data and c o n d i t i o n s f o r data c o l l e c t i o n .  C44H28ClInN4  Fw  763.015  Space Group: P2 1 /n  Vol  3432.5 A  a = 10.099(1) A  Z  4  b = 16.117(2) c = 21.090(2)  1.476 g cm Dc -3 D 0 a - 1 . 4 8 ( 1 ) g cm  B = 90.70(1)°  v  3  -3  = 7 . 2 2 cm"1  Radiation:  Mo K a , g r a p h i t e monochromator  Scan t y p e :  u - 2e  Scan range:  (0.47 + 0 . 3 5 t a n e ) ° i n u, extended 25** f o r each background measurement  Scan speed:  0.75 to 10.1 deg m i n " 1 , to g i v e I / o ( I ) ^ 2 0  Aperture:  1.33 x 4 mm, 173 mm from c r y s t a l  Standards:  190, 6 4 1 , 05T2, 6 0 4 ; measured every hour o f exposure time  Data c o l l e c t e d :  + h k i f o r 2e <_ 5 5 ° ; 7859 r e f l e c t i o n s  o(I):  {Int + 4(DG) + (0.041 J 2 }* 5 ; I n t i s i n t e g r a t e d peak c o u n t , BG i s the sum o f the background counts and 1 i s the i n t e n s i t y  a  by n e u t r a l buoyancy i n  CCI4  and CH2C1 2  -68-  T a b l e  4.2.  I t CW  F i n a l  a t o m i c  p o s i t i o n a l  T  t  and  thermal  parameters  o i i  1  b  022  033  ta  3 » 9 3 . 1 ( 3)  1 7 9 7 . 0 ( 2)  1 5 1 6 . 3 ( 1)  • .23(2)  3.3« (2)  2.6»(2)  Cl  1387(  1)  2388 ( D  1 7 0 6 ( 1)  S.2(  6 . 1 ( 1)  5.5(  1(1)  328K ( •>  • 9 9 ( 3)  17»9< 2)  5.6(  2J  3. • ( 2)  • (•)  3522 ( •)  1 3 6 9 { 3)  S«B( 2)  5.5(  3|  3.5(  • O)  »761( «)  2739(  3)  1 2 0 2 ( 2)  5.1(  2)  1(2)  • 5791 •)  1 8 9 1 ( 3)  2«01( 2)  «.«{  2)  C(D  28U» ( 5)  -119(  3)  13«1< 2)  «.8(  3)  C(2)  2 6 6 5 1 6)  - 8 6 3 ( 3)  1 7 0 3 ( 3)  6.91  •)  CCD  3 0 i e ( 6)  - 7 0 3 ( 3) 150 ( 3)  1»  0.81  3.0(  2)  -0.»(  2)  - 0 . 3 ( 2)  0.2(  2)  2)  2.8(  2)  -0.3(  2)  -0.3(  2)  0.0(  2)  •. 0 (  2)  3 . 1 ( 21 - 0 . 7 ( 2)  -0.3(  2)  0.2(  2)  3.B(  2)  3.1(  2) - 0 . 8 (  2)  -0.6(  2)  0.5(  2)  3 . • ( 3)  «.K  -0.5(  2)  -0.3(  2|  -0.0(  2)  3.7(  3)  • • * ( 3) - 1 . 0 (  3)  -0.9(  3)  0.6(  2)  3)  -0.8(  3)  1-0(  2)  7.0(  •)  •.1(  3)  • .0(  2 3 U 6 ( 2)  5.2(  3)  3.7(  3)  3.2(  2)  -0.2(  2)  -0.«  2) 0 . 5 (  2)  2) - 0 . 6 (  2)  -0.2(  2)  0.7(  2)  2)  -0.6(  2)  - 0 . » ( 2)  0.6(  2)  -0.9(  3)  - 1 . 0 ( 2)  0.9(  2)  3 9 0 9 ( 5)  C (6)  tt u u 5 ( 5)  C(T)  • 976 (  C(8)  5«23( 5)  C(9)  5 1 9 9 ( 5)  263 1 ( 3)  2619(  2)  • .3(  3)  ».M  C(10)  5 5 6 1 ( 5)  3 2 7 7 ( 3)  2 2«7( 2)  • .7(  3)  C(U)  5 3 9 2 ( 5)  C(12)  5890 (  C(11)  5 5 7 5 ( 6)  3)  2 8 8 6 < 2)  • .5(  3)  3.9(  3)  3.6(  1 36 1 ( 3>  2 9 0 9 ( 2)  S.0(  3)  • • 1 ( 3)  3.1(  3 * 6 2 ) 2)  6.7(  3)  5 . 1 ( 3)  3.1(  2)  2510 ( • )  3 2 8 6 ( 2)  5.8(  3)  5.5(  3.5{  3) - 1 . « (  3)  -0.9(  2)  0.»< 2)  3)  3.2(  2) - 0 . & 1  2)  -0.7(  2)  0.2(  ».S< 3)  3.5(  2) - 0 . 7 (  2)  - 0 . 7 ( 2)  O.u( 21  2)  O.M  2)  1590 ( 2)  « . 9 ( 31  » . » ( 3J  3.7(  2)  -1.21  2)  «02« ( •)  1207 { 2)  6.5(  5.1(  ».M  3)  -2.2(  3)  -0.7(  2)  0.6(  3)  5 . 2 ( 3)  3.9(  3) - 2 . 0 (  3)  - 0 . 1 ( 2)  0.9(  2)  2)  -0.«(  2)  0 . 0 ( 2)  0.9(  2)  2)  -0.3(  2)  0 . 1 ( 2)  O.M  2)  0.0(  2)  O.M  2)  3946 (  •)  3 0 7 7 ( 3)  C(15|  « 39 3 ( 5)  2 6 6 7 ( 3)  3390(  C(17) C<1S»  *)  1 8 8 5 ( 3) 1«50 ( • )  597(  2)  « . 1 ( 3)  3)  2)  ».7{  3)  « . 0 ( 3)  • 8 1 2)  «.7(  3)  • . 3 ( 3)  3.1(  589(  6.9(  - S 3 » < 2)  3)  - 3 S S C 2)  2 9 6 6 ( 5)  6 6 3 ( 3)  32 1 ( 2 | 688  (  «)  3.1(  2)  • . » ( 3)  2.9[  2)  -0.1(  3)  -0.11  2)  0.3(  2)  3)  3.1(  2) - 0 . 7 (  2)  -0.5(  2)  -O.M  2)  3)  3.1(  2) - 0 . 2 (  2)  -0.3(  2|  -O.M  2)  2)  -0.3(  2)  -0.71  2)  * . » < 3) S.0(  3>  « . 7 ( 3J  2)  0 . 1 ( 2)  3)  «.0(  « . 9 ( 3)  33 ( 2)  711(  - S O ( 3)  •)  3.1(  2896 < M  2 6 6 5 1 5)  C(20|  3.9(  ! - » < 3)  3.6(  -0.»(  2)  « E s t i m a t e d standard d e l a t i o n s In t h i s . n d o t h e r t a b l e s a r t , 1 , e n In parentheses and correspond t o the l e a s t j i g n U U a n t The p o s i t i o n a l par i n t e r s have been m u l t i p l i e d bjr 1 0 * and the thermal parameters bjr 1 0 * b  U  U  • «ij/(2. it*«j*) A z  2  -  2)  3)  3358(  5)  3 7 8 3 ( 5)  3)  -0.5(  • S S b  C(H|  (  •)  1760(  C{1«)  (  - 0 . 5 ( 1)  3)  2310{  C(5)  *)  3)  1) - 0 . 1 ( H  3) - 0 . 8 (  3 3 9 5 1 5)  *)  0 . 20 (1)  •- 0 . 3 3 ( 1 )  -0.39(2)  1)  C(«)  5o0(  023  013  012  The thermal e l l i p s o i d Is g'ven b / e x p [ - ( e , , n  ?  • ^  * 8,3'  • Z ^ n k • 2e, «i • 2 e j k i ) ] . 3  2  dtoUs.  -69-  Table 4 . 3 . Group 1C1 2C1 3C1 4C1  -  1C6 2C6 3C6 4C6  Group 1 Cl 2C1 3C1 4C1 a  -  x c 0.1566(2) 0.4074(3) 0.7040(3) 0.4835(3)  R B  1C6 2C6 3C6 4C6  R i g i d group parameters.  •>  l 3.38(9) 3.38(9) 3.37(9) 3.7(1)  y  c  -0.1402(2) -0.0409(2) 0.4590(2) 0.3297(2) B2 4.5(1) 4.6(1)  i:if!l  c -0.0041(1 ) 0.4037(1) 0.2875(1) -0.1213(1)  6  c  n  1.430(3) -1.642(3) -2.114(3) -1.899(3)  -2.662(2) 3.017(2) 2.997(3) -3.100(2)  0.666(3) -2.137(3) -0.457(3) 1.245(3)  B3 5.3(1) 5.1(1) 4.9(1 6.3(2)  *4 4.8(1) 5.KD 5.2(1) 6.2(2)  B5 4.7(1) 5.0(1) 5.6(1) 6.3(2)  z  See S . J . LaPlace and J . A. I b e r s , Acta C r y s t a l l o g r . 18, 511 (1965) f o r d e f i n i t i o n o f group parameters.  k B n i s the temperature f a c t o r o f atom C n i n the phenyl r i n g i n A^.  B6  4.KD  4.3(1) 4.7(1) 5.5(1)  Table 4 . 4 .  Derived Hydrogen atom p o s i t i o n a l and thermal  parameters 3  O (A*)  z  z  y  BC ( 1 6 )  255  30  -63  5.9  HC(17)  346  165  -96  6.2  BC(13)  579  417  24  6.3  HC(12)  635  450  136  6.5  HC(8)  582  291  356  6.2  HC ( 7 )  501  154  3  H C (3)  302  HC(2)  Itoi  ea  6.2  -109  265  6.3  235  -139  154  6.3  H1C2  28  -70  70  7.0  B1C3  -64  -179  11  6.3  64  -249  -64  7.3  B1C5  285  -210  -79  7.2  B1C6  377  -101  -19  6.5  H4C2  276  349  -75  6.8  H4C3  312  392  -180  7.5  B4C4  519  373  -226  7.9  B4CS  691  311  -167  8.3  B4C6  655  268  -63  7.3  B3C2  475  471  270  7.9  B3C3  590  576  323  7. 4  B3C4  819  565  340  9. 1  B3C5  933  448  305  9.2  B3C6  8 18  342  252  B2C2  200  H2C3  219  B2C4  • 27  B2C5 B2C6  B 1 CU  6  463  -118  497  615  -88  439  596  -11  344  The p o s i t i o n a l parameters have been m u l t i p l i e d by 10  2  7. 1  369  -70  thermal parameters by 10 .  8.3  7.8 7.7 7.6 6.7  and the  -71Table 4 . 5 . In In In In In  -  S e l e c t e d Interatomic d i s t a n c e s ( A ) and angles (deg). Distances  N O )  2 . 1 6 0 ( 4 )  C(5)  -  C(6)  1.400(7)  N(2)  2 . 1 5 8 ( 4 )  C(6)  -  C(7)  1.430(7)  N  2 . 1 5 8 ( 4  C(7)  -  C(8)  1.344(7)  N(4)  2 . 1 4 6 ( 4 )  C(B)  -  C(9)  1.431(6)  Ct  2 . 3 6 9 ( 2 )  C(9)  -  C(10)  1.399(7)  C O O )  3  N O )  -  N ( 3 )  4 . 1 5 0 ( 6 )  N(2)  -  N ( 4 )  4 . 1 1 6 ( 5 )  N O )  -  C(l)  1 . 3 8 6 ( 6 )  N O )  -  C ( 4 )  1 . 3 8 3 ( 6 )  N(2)  -  C ( 6 )  N(2)  -  C ( 9 )  N(3)  -  N(3) N(4) N(4) C O ) C(2) C(3) C(4)  1.403(7)  -  C(11)  -  C ( 1 3 )  1.354(7)  C(13)  -  C ( 1 4 )  1.436(7)  1 . 3 7 8 ( 6  C(14)  -  C(15>  1.394(7)  1 . 3 8 0 ( 6 )  C(15)  -  C ( 1 6 )  1.403(7)  C(ll)  1 . 3 8 0 ( 6 )  C(16)  -  C(17)  1.437(7)  -  C ( 1 4 )  1 . 3 7 8 ( 6 )  C(17)  -  C ( 1 8 )  1.344(7)  -  C  16)  1 . 3 7 7 ( 6 )  C 0 8 )  -  C ( 1 9 )  1.437(6)  -  C ( 1 9 )  1 . 3 7 2 ( 6 )  C(19)  -  C ( 2 0 )  1.412(7)  1 . 4 3 4 ( 7 )  C(20)  -  C O )  1.393(7)  Jill!  1.438(7)  -  C ( 3  1 . 3 4 9 ( 7 )  C(20)  -  1 Cl  1.506(6)  -  C ( 4 )  1 . 4 2 8 ( 7 )  C(5)  -  2C1  1 . 4 9 £ ( 6 )  -  C ( 5 )  1 . 4 1 1 ( 7 )  C O O )  -  3C1  1.502(7)  C 0 5 )  -  4C1  1.507(6)  Anql es  N O )  8 5 . 5 ( 2 )  1C1  C(20)  -  C(19)  115.5(2)  N(2)  8 5 . 2 ( 1 )  1C1  C(20)  -  C O )  118.4(2)  N(3)  8 5 . 4 ( 2 )  2C1  C(5)  -  C(4)  116.1(2)  N(4)  8 5 . 5 ( 2 )  2C1  C(5)  -  C(6)  1 1 7 . 0 ( 2 )  1 4 7 . 9 ( 2 )  3C1  C(10)  -  C(9)  1 1 6 . 3 ( 2 )  1 4 6 . 3 ( 2 )  3C1  C(10)  -  C O D  1 1 7 . 4 ( 3 )  1 0 5 . 1 ( 1 )  4C1  C 0 5 )  -  C(14)  H E . 8 ( 2 )  1 0 5 . 9 ( 1 )  4C1  C(15)  -  C(16)  1 1 5 . 0 ( 2 )  1 0 6 . 9 ( 1 )  C O )  C(2)  -  C(3)  1 0 8 . 1 ( 5 )  1 0 7 . 8 ( 1 )  C(2)  C(3)  -  C(4)  1 0 7 . 6 ( 4 )  1 2 5 . 9 ( 3 )  C(3  C(4)  -  C(5)  1 2 6 . 2 ( 4 )  1 2 6 . 4 ( 3 )  C(4)  C(5)  -  C(6)  126.7(4)  1 2 5 . 1 ( 3 )  C(5)  C(6)  -  C(7)  1 2 5 . 6 ( 4 )  1 2 4 . 7 ( 3 )  C(6)  C(7)  -  C(8)  1 0 7 . 7 ( 4 )  1 2 5 . 6 ( 3 )  C(7)  C(8)  -  C(9)  1 0 8 . 2 ( 5 )  1 2 5 . 9 ( 3 )  C(8)  C(9)  -  C(10)  126.1(5)  1 2 5 . 4 ( 3 )  C(9)  C O O )  -  1 2 6 . 2 ( 5 )  125.6  C O O )  C O D  -  C O D C  1 2 5 . 4 ( 5 )  C(12)  -  C(13)  107.2(5)  C(13)  -  C(14)  107.8(4)  C(14)  -  C(15)  125.6(4)  C(15)  -  C  126.0(4)  C(16)  -  C(17)  •  N O ) N(2) Ct Ct Ct Ct  In In In In In In In In  1 2 5 . 7 ( 4 )  N ( l )  1 2 5 . 9  2)  N(3) N(4)  4)  1 2 5 . 9 ( 4  N(2) N ( 3 )  : -  C ( 1 6 )  : -  N(4)  1 2 5 . 5  1 2 5 . 5 ( 4 1 2 6 . 2 ( 4 1 2 5 . 8 ( 4 ) 1 0 8 . 9  urn  4  1 0 8 . 4 ( 4  N(2)  1 0 8 . 0 ( 4  N(2)  1 0 9 . 0 ( 4  N ( 3 )  1 0 8 . 8  N  108.6(4)  3  N M )  COB)  1?)  C03) 14)  4  1 0 8 . 2 ( 4 )  N O )  *U)  11)  1 2 4 . 7 ( 5 )  N O ) N  3)  4  1 0 8 . 8 ( 4 )  15) C(16) C C(19) C(20)  12)  16)  C O D  125.1(4)  C(18)  107.6(4)  : ci!Si: : :  C(19)  107.6(4)  C(20)  125.2(4)  C O )  126.0(4)  C(2)  126.0(5)  -72Table  4.5,  N N a b  -a -d -b -c c - d a -m d -m  a N N a b  -  N a d b c  -  d b c c d  continued. Pyrrole  pyrrole  4  A v e r a g e  Pyrrole 1  Pyrrole  1.386(6) 1.383(6) 1.434(7) 1 349(6) 1.428 6 1.393(7) 1.411(7)  1.377(6) 1.378(6) 1.437(6) 1.344(7) 1.437(7) 1.403(7) 1.412(7)  1.380(6) 1.378(6) 1.438(7) 1.354(7) 1.436(7) 1.403(7) 1.394(7)  1.378(6) 1.380(6) 1.430(7) 1.344(7) 1.431 6) 1.400(7) 1.399(7)  1.380(4) 1.380(2) 1.435(4) 1.348(5) 1.433(4) 1 .402(7)  107.1(4) 108.2(4) 108.9(4) 108.1(5) 107.6(4)  107.3(1) 108.6(4) 108.8(4) 107.6(4) 107.6(4)  107.2(4) 109.0(4) 108.8(4) 107.2(5) 107.8(4)  107.7(4) 108.4(4) 108.0(4) 107.7(4) 108.2(5)  107.3(3) 108.5(3) 108.6(4) 107.6(4) 107.8(3)  2  3  Table 4 . 6 .  S e l e c t e d planes o f the p o r p h y r i n m a c r o c y c l i c s k e l e t o n  Plane #1:  - 0 . 8 9 8 3 x + 0.4132y + 0.1491z + 2.0711 = 0 a  Plane #2:  -0.9052x + 0.4003y + 0.1426z + 2.2543 = 0 o  P e r p e n d i c u l a r displacements  Plane #2  Plane #1 In ci . N(D* N(2)* NO)* N(4) C(D C(2) C(3) C(4)  C(5) C(6) C(7) C(8) C(9)  3  *  0.6104(3) 2. 978 (2) 0. 014 (4) - 0 . 014 (4) 0. 015 (4) - 0 . 014 (4) - 0 . .136 (5) - 0 . .347 (6) - 0 . .356 (6) - 0 . .117 (5) - 0 , .128 (5) - 0 .074 (5) - 0 , .103 (6) - 0 . 0 6 8 (6) - 0 .029 (5)  (A)  C(10) COD C(12) C(13) C(14) C(15) C(16) C(17) C(18) C(19) C(20) 1C1 2C1 3C1 4C1  -0.052(5) -0.049(5) -0.185(6) -0.224(6) -0.086(5) -0.122(5) -0.095(5) -0.219(6) -0.204(5) -0.089(5) -0.141(5) -0.241(3) -0.327(3) -0.163(3) -0.310(3)  In Cl NO)* N(2)* N(3)* N(4)=*  CO)*. C(2)* C(3)* C(4)* C(5)* C(6)*  C(7)*  C(8)* C(9)*  The plane equations are i n terms o f orthogonal  0.7115(3) 3.079 (2) 0.141 (4) 0.066 (4) 0.091 (4) 0.108 (4) 0.012 (5) - 0 . 1 8 7 (6) -0.211 (6) 0.007 (5) -0.023(3) 0.011 (5) - 0 . 0 3 8 (6) - 0 . 0 1 9 (6) 0.028 (5)  c o o r d i n a t e s i n A.  I n d i c a t e s the atoms i n c l u d e d i n the c a l c u l a t i o n of the mean p l a n e .  C(10)* c(n)*  C(12)* C(13)* C(14)* C(15)* C(16)* C(17)* C(18)* C(19) C(20)* 1C1 2C1 3C1 4C1  -0.006  (5)  0.006  (5)  - 0 . 1 4 2 (6) - 0 . 1 6 8 (6) - 0 . 0 0 8 (5) - 0 . 0 2 5 (5) 0.023 (5) - 0 . 0 8 2 (6) -0.051 (6) 0.055 (5) 0.015 (5) -0.061 (3) - 0 . 2 2 0 (4) -0.141 (4) -0.214 (4)  Table 4.7. Metal Bi Zr Hf Fe Tl In Fe (VO) Mn Fe Fe Fe Fe Fe Zn Zn Zn Fe Fe Fe Mn Mg Mn Co Co  Representative  Oxidation State  Ionic b Radius  III IV IV II III III II (ID III III III II III III II II II III II III III II III II II  0.96 A 0.79 0.78 0.74 0.95 0.81 0.74 0.66 0.64 0.64 0.74 0.64 0.64 0.74 0.74 0.74 0.64 0.74 0.64 0.66 0.66 0.66 0.72 0.72  metalloporphyrins  w i t h s i g n i f i c a n t out-of-plane Coordination Number  Metalloporphyrin Bi(OEP) (0Ac) Zr(0EP) (0Ac) Hf(0EP) deoxyhemoglobin (horse) ClTl(OEP) ClIn(TPP) deoxyhemoglobin (human) OV(OEP) (l-Melm)Mn(TPP) 0[Fe(TPP)] ClFe(proto-IX) (2-MeIm)Fe(TPP) (2-MeIm)Fe(TpivPP) ClFe(TPP) (C10<,)Zn(TPP) (py)Zn(TPyP) (py)Zn(OEP) methemoglobin (horse) deoxy-erythrocruorin metmyoglobin (whale) CLMn(TPP) (H 0)Mg(TPP) N Mn(TPP) (l-Melm)Co(OEP) (l-Melm)Co(TPP) 2  2  2  2  3  5 8 8 (6) 5 5 (6) 4 5 5 5 5 5 5 5 5 5 (6) (6) (6) 5 5 5 5 5  Metal Ion  displacement  Q  Displacement 1.09 A 1.02 1.01 0.75 0.69 0.61  (M-N)  ave  of the metal i o n . Core j Radius  a  Doming  Reference  2.32 A 2.047 A 0.13 A 2.268,2.259 2.024,2.014 0.17,0.21 2.257 2.016 0.13  f  2.212 , 2.156 0.60(a);0.63(8) 2.1(aorB) 0.54 2.101 0.52 2.128 0.50 2.087 0.48 2.062 0.42 2.086 0.40 2.072 0.38 2.049 0.35 2.076 0.33 2.073 0.31 2.067 M).3 M).3 0.27 2.04 0.27 2.008 0.27 2.072 0.23 2.005 0.13 1.955 0.13 1.977  2.10 2.067 2.008 2.030 2.065 2.027 2.007 2.044 2.033 2.046 2.047 2.043  f  f  2.00*> 1.989 2.055 1.992 1.950 1.973  114 115 115 62 0.06 116 0.10 T h i s work <o, 22(o);0.17 (8) 74 . 0.06 117 0.04 66 0.04 48 0.06 41 0.13 64,65 0.03 118 40 0.09 119 0.04 21 0.09 120 28 25 0.13 95 0.0 121 122 123 0.03 55 0.01 54  A b b r e v i a t i o n s : OAc, a c e t a t e ; Me, methyl; py, p y r i d i n e ; OEP, o c t a e t h y l p o r p h y r i n d i a n i o n ; TPP, t e t r a p h e n y l p o r p h y r i n d i a n i o n ; proto-IX,  protoporphyrin  IX d i a n i o n ; TpyP, t e t r a ( 4 - p y r i d y l ) p o r p h y r i n d i a n i o n .  from r e f e r e n c e 8. p e r p e n d i c u l a r displacement  o f the metal i o n from the mean plane o f the four p y r r o l e n i t r o g e n s .  average d i s t a n c e from the c e n t e r o f the plane o f the f o u r p y r r o l e n i t r o g e n s to a p y r r o l e n i t r o g e n . d i f f e r e n c e between the displacement o f the m e t a l from the mean plane o f the four p y r r o l e n i t r o g e n s and t h e mean plane o f the 24-atom p o r p h y r i n s k e l e t o n . g  displacement  i n t h i s case i s measured from the mean plane o f the 24-atom p o r p h y r i n  t h i s d i s t a n c e was c o n t r a l n e d t o t h i s v a l u e  i n refining  the atomic p o s i t i o n s .  skeleton.  -75-  PART TWO  PERTURBED ANGULAR CORRELATION STUDY ON MYOGLOBIN ( R a d i o a c t i v e "'""'""'"In-labelled p o r p h y r i n / R e c o n s t i t u t i o n / x determination) c  -76-  CHAPTER V PERTURBED ANGULAR CORRELATIONS General I n t r o d u c t i o n D u r i n g t h e past decade, s e v e r a l l a b e l l i n g t e c h n i q u e s have been developed motions,  t o determine  the r o t a t i o n a l c o r r e l a t i o n t i m e s , i n t e r n a l  and c o n f o r m a t i o n a l changes i n b i o l o g i c a l macromolecules.  As  one example, f l u o r e s c e n c e d e p o l a r i z a t i o n measurements can p r o v i d e a meas u r e o f the r o t a t i o n a l c o r r e l a t i o n time o f a s m a l l chromophore bound t o a macromolecule (127).  R o t a t i o n a l c o r r e l a t i o n times can a l s o be o b t a i n e d  from magnetic r e l a x a t i o n times ( 1 2 8 ) , and from ESR l i n e - s h a p e a n a l y s i s (129) i s o n l y i n v e r y r e c e n t y e a r s t h a t the method o f p e r t u r b e d a n g u l a r c o r r e l a t i o n s (P.A.C.) has been u t i l i z e d as a l a b e l l i n g t e c h n i q u e i n t h e study o f b i o l o g i c a l macromolecules.  S e v e r a l p u b l i c a t i o n s have d i s c u s s e d  how P.A.C. measurements can g i v e m o t i o n a l and s t r u c t u r a l i n f o r m a t i o n about b i o l o g i c a l macromolecules The  (7-12).  i n f o r m a t i o n gained from P.A.C. measurements i s o f t e n s i m i l a r t o  t h a t o b t a i n e d i n n u c l e a r and paramagnetic s t u d i e s b u t , i n some cases not o b t a i n a b l e i n any o t h e r way.NMR i s a r e l a t i v e l y an i n s e n s i t i v e  technique,  e s p e c i a l l y i n t h e case o f v e r y d i l u t e samples, which i s o f t e n t h e case f o r -4  -5  b i o l o g i c a l macromolecules (10 -10  M).  T h i s d i f f i c u l t y a r i s e s from t h e  weakness o f an n.m.r. s i g n a l compared w i t h t h e background n o i s e o f t h e i n s t r u m e n t s used.  F o r proton-n.m.r. o f b i o l o g i c a l macromolecules i n  which aqueous s o l v e n t s a r e r e q u i r e d , i t i s n e c e s s a r y i n D2O r a t h e r than i n w a t e r .  t o prepare  t h e sample  T h i s i s t o e l i m i n a t e the s t r o n g H^O-proton  resonance t h a t obscures most o f the resonances o f p r o t o n s o f i n t e r e s t .  -77-  Moreover, i n t e r p r e t a t i o n o f n.m.r. s p e c t r a f o r l a r g e macromolecules i s r a r e l y simple.  The f l u o r e s c e n c e d e p o l a r i z a t i o n t e c h n i q u e  o p t i c a l transparency p o t e n t i a l use.  f o r o p e r a t i o n which thus g r e a t l y l i m i t s i t s  Both n.m.r. and e s r t e c h n i q u e s  ment f o r o p e r a t i o n .  requires  demand expensive  equip-  The P.A.C. method has t h e advantage o f b e i n g  a p p l i c a b l e t o s o l u t i o n s and s o l i d s w h i c h opens t h e p o s s i b i l i t y f o r in v i v o experimentation.  The s i m p l i c i t y o f e x p e r i m e n t a l  measurements,  -12 w i t h c o n c e n t r a t i o n s e n s i t i v i t y a p p r o a c h i n g 10 f a c t that the t h e o r e t i c a l understanding  M, t o g e t h e r w i t h t h e  of the e f f e c t s of molecular  m o t i o n on a n g u l a r c o r r e l a t i o n s has now become more complete, make P.A.C. a p o t e n t i a l l y u s e f u l l a b e l l i n g technique.  D e s p i t e i t s obvious p o t e n t i a l ,  r e l a t i v e l y few P.A.C. s t u d i e s have been r e p o r t e d . i s probably  The p a u c i t y o f d a t a  a t t r i b u t a b l e to the l a c k of v e r s a t i l i t y i n s e l e c t i v e attach-  ment o f r a d i o a c t i v e r o t a t i o n a l l a b e l s t o s p e c i f i c s i t e s on macromolecules. T h i s t h e s i s demonstrates t h a t "^"'"In-labelled p o r p h y r i n s c a n be v e r y s e l e c t i v e l y incorporated i n t o myoglobin. P.A.C. experiments r e q u i r e t h e i n c o r p o r a t i o n o f a gamma e m i t t e r i n t o the m o l e c u l e s under study. l l l m  Cd(t  =49 m i n ) ,  1  6 2  'i  ^ ^ I n ( t , =2.8 d a y s ) . 's  S e v e r a l i s o t o p e s (gamma e m i t t e r s ) can be used:  Zn(t,=9h),  1 1 9 m  Hg(t  "2  =43 m i n ) , ° Pb(t,=68 m i n ) , and 2  ]  4  -3  "5  I t i s obvious that ^"^In w i t h i t s convenient  l i f e , 2.8 days i s t h e u s e f u l i s o t o p e from t h e p r a c t i c a l  half-  viewpoint.  F u r t h e r m o r e , i n d i u m - I l l produces two gamma r a y s i n s u c c e s s i o n , each w i t h a convenient first  energy f o r d e t e c t i o n .  M a r s h a l l e t a l . , (13,14) were t h e  t o show how gamma-gamma c o i n c i d e n c e measurements f o r t h i s type o f  energy cascade can g i v e d i r e c t i n f o r m a t i o n about c h e m i c a l bonding and motional  flexibility  The  a t an i n d i u m - l a b e l l e d s i t e on a macromolecule.  r a d i o a c t i v e l a b e l s can be made s p e c i f i c by b i n d i n g t h e r a d i o a c t i v e  -78-  nuclei f i r s t can be bound  t o some c h e m i c a l complexes. to  For i n s t a n c e , the Cd i s o t o p e  a c h e m i c a l complexing agent such as EDTA which i s  c o v a l e n t l y a t t a c h e d t o an a c t i v e group such as a s u l f y d r y l reagent. T h i s p a r t o f the t h e s i s d e s c r i b e s the p r e p a r a t i o n o f i n d i u m - I l l meso-protoporphyrin  labelled  IX, and how P.A.C. measurements can g i v e r o t a t i o n a l  c o r r e l a t i o n time of myoglobin.  The present r e c o n s t i t u t i o n of i n d i u m - I l l  meso-protoporphyrin IX i n t o apomyoglobin  (myoglobin w i t h i t s n a t i v e heme  removed) r e p r e s e n t s the f i r s t m o t i o n a l probe l o c a t e d a t the metal c e n t e r of  the a c t i v e s i t e on a p r o t e i n .  time o f myoglobin  The r o t a t i o n a l  correlation  o b t a i n e d by P.A.C. method i s 16 nsec a t 12°C. The  d i s c r e p a n c y between the e x p e r i m e n t a l T^* and the  calculated  from  Debye's model i s p r o b a b l y due t o the n o n - s p h e r i c a l c o n f o r m a t i o n of myoglobin.  *x  c  i s used t o denote  the r o t a t i o n a l c o r r e l a t i o n  time,  -79CHAPTER VI  THEORY OF PERTURBED ANGULAR CORRELATIONS OF NUCLEAR RADIATION  6.1  Introduction For decades p h y s i c i s t s  d e t e r m i n a t i o n of p r o p e r t i e s of  radiations emitted,  It  is  have employed of e x c i t e d  and of  technique  for  the  n u c l e a r s t a t e s and m u l t i p o l a r i t i e s  interactions  only w i t h i n t h i s decade that  the P . A . C .  responsible  f o r the  emission.  chemists began to use P . A . C .  measure-  ments to o b t a i n m o t i o n a l and s t r u c t u r a l i n f o r m a t i o n on b i o l o g i c a l macromolecules. The p r o b a b i l i t y W ( 6 ) f o r e m i s s i o n the a n g l e 8 between the emitted  r a d i a t i o n and some f i x e d d i r e c t i o n .  total  r a d i a t i o n from a r a d i o a c t i v e  spins  a r e randomly o r i e n t e d  for  the e m i s s i o n  can be observed  of a n u c l e a r r a d i a t i o n depends on  sample  is  isotropic  i n space s i n c e t h e r e  of the r a d i a t i o n .  if  the  The  nuclear  i s no p r e f e r r e d d i r e c t i o n  An a n i s o t r o p i c  p a t t e r n of  o n l y from an ensemble of n u c l e i that  emission  a r e not randomly  oriented. One method of o b t a i n i n g o r i e n t e d n u c l e i s p i n system to a v e r y low t e m p e r a t u r e . orientation orientations  of  in  field  Another method i s  the c a s e o f P . A . C .  successive emission emit  the  o c c u r s w i t h the d i s t r i b u t i o n of  or an e l e c t r i c  first  If  field  spin  A p p l i c a t i o n of a v e r y  gradient w i l l also a l i g n  the s e l e c t i o n of n u c l e i w i t h a l i g n e d s p i n s the n u c l e i such as i n d i u m - I l l , decay  o f two r a d i a t i o n s ,  the as  through  c h o o s i n g o n l y those n u c l e i which  r a d i a t i o n i n a given d i r e c t i o n i s  n u c l e i whose s p i n s  nuclear  At thermal e q u i l i b r i u m , a net  a c c o r d i n g to the Boltzman f u n c t i o n .  s t r o n g magnetic spins.  the s p i n - s y s t e m  i s by c o o l i n g the  are e s s e n t i a l l y aligned  equivalent  i n that  to  direction.  selecting The  s u c c e e d i n g second  r a d i a t i o n then shows a d e f i n i t e a n g u l a r dependence  with respect to the d i r e c t i o n of the f i r s t r a d i a t i o n . There i s a s t r o n g a n g u l a r c o r r e l a t i o n between t h e d i r e c t i o n s o f p r o p a g a t i o n o f t h e gamma r a y s i n cascade.  I t i s t h i s property  that  enables the P.A.C. method t o m o n i t o r m o l e c u l a r motion. F o l l o w i n g t h e e m i s s i o n o f t h e f i r s t gamma r a y , t h e a n g u l a r c o r r e l a t i o n , W ( 0 ) w i l l be s t r o n g l y p e r t u r b e d i f t h e o r i e n t a t i o n o f t h e s p i n o f t h e n u c l e u s i n the i n t e r m e d i a t e s t a t e changes by i n t e r a c t i o n w i t h i t s s u r r o u n d i n g s . I n the s e m i - c l a s s i c a l p i c t u r e , these i n t e r a c t i o n s produce a p r e c e s s i o n o f the n u c l e i around t h e symmetry a x i s . The change i n n u c l e a r o r i e n t a t i o n produces an a l t e r e d a n g u l a r c o r r e l a t i o n . observed  S e v e r a l workers  (7,8,9)  t h a t t h e a n g u l a r c o r r e l a t i o n o f t h e gamma r a y cascade  the decay o f ^ ^ ^ I n i s s t r o n g l y  p e r t u r b e d when t h e r a d i o a c t i v e  bound t o a macromolecule i n aqueous s o l u t i o n . anisotropic  have  following ion i s  I n o r d e r t o observe an  c o r r e l a t i o n i n t h e absence o f an a p p l i e d  f i e l d , the i n t e r -  mediate s t a t e much have n u c l e a r s p i n _> 1, so t h a t t h e n u c l e u s may possess a quadrupole moment.  The q u a d r u p o l a r  r u p o l e moment w i t h e x t e r n a l the a n g u l a r c o r r e l a t i o n . for  i n t e r a c t i o n o f t h e n u c l e a r quad-  e l e c t r i c f i e l d gradient i s the basis of  T h i s i n t e r a c t i o n t u r n s out t o be an advantage  t h e s t u d y o f m o l e c u l a r r o t a t i o n a l m o t i o n , because t h e n u c l e a r s p i n  o r i e n t a t i o n r a t e due t o a q u a d r u p o l a r  i n t e r a c t i o n i s a f f e c t e d by m o l e c u l a r  r o t a t i o n a l m o t i o n , b u t n o t by r e l a t i v e t r a n s l a t i o n a l m o t i o n . By t h e study o f t h e p e r t u r b e d a n g u l a r c o r r e l a t i o n o f gamma r a d i a t i o n from a r a p i d l y r e o r i e n t i n g r a d i o a c t i v e n u c l e u s , t h e n u c l e a r r e l a x a t i o n t i m e c a n be measured.  S i n c e t h e n u c l e a r r e l a x a t i o n time depends  on t h e r a t e o f r o t a t i o n o f t h e m o l e c u l e  t o which t h e r a d i o a c t i v e  strongly nucleus  i s bound, i t may be used t o e s t i m a t e t h e r o t a t i o n a l c o r r e l a t i o n t i m e , T  c  w h i c h i s a measure of t h e time i t t a k e s f o r t h e m o l e c u l e t o change i t s o r i e n t a t i o n by the o r d e r of one r a d i a n .  6.2  Theoretical The  consideration  t h e o r y o f a n g u l a r c o r r e l a t i o n i s p r o b a b l y one o f t h e b e s t and  most comprehensive t h e o r i e s on n u c l e a r only the theory of extranuclear w i l l be c o n s i d e r e d .  phenomena.  perturbations  For the present case,  on a n g u l a r c o r r e l a t i o n s  An e x c e l l e n t r e v i e w o f t h e t h e o r y o f p e r t u r b e d  a n g u l a r c o r r e l a t i o n s i s a v a i l a b l e i n t h e a r t i c l e by F r a u e n f e l d e r Steffen  and  (130).  It i s f e l t w i l l provide  that a b r i e f d i s c u s s i o n of fluorescence  depolarization  a b e t t e r u n d e r s t a n d i n g t o P.A.C. because c o n c e p t u a l l y and  instrumentally  i t i s analogous t o P.A.C.  In fluorescence  d e p o l a r i z a t i o n , i f the e x c i t i n g r a d i a t i o n (usually  i n the u l t r a - v i o l e t region) i s p o l a r i z e d , the p r o b a b i l i t y of absorption w i l l depend on t h e o r i e n t a t i o n o f t h e m o l e c u l e , b e i n g maximum i f t h e d i r e c t i o n o f t h e d i p o l e moment change i s p a r a l l e l t o t h e d i r e c t i o n o f propagation. (^10  I f , w i t h i n the l i f e t i m e of the e x c i t e d e l e c t r o n i c s t a t e  s e c ) , t h e m o l e c u l e s do n o t r o t a t e a p p r e c i a b l y ,  r a d i a t i o n w i l l a l s o be h i g h l y p o l a r i z e d . however, r o t a t i o n a l movement o c c u r s .  then t h e f l u o r e s c e n t  I n a s o l u t i o n of molecules,  S i n c e t h i s i s a random p r o c e s s ,  i t l e a d s t o r a n d o m i z a t i o n o f t h e o r i e n t a t i o n o f t h e d i p o l e moments i n the time between a b s o r p t i o n  and e m i s s i o n ,  of the f l u o r e s c e n t r a d i a t i o n .  which r e s u l t s i n d e p o l a r i z a t i o n  T h i s means t h a t f l u o r e s c e n c e  depolarization  can be used t o measure t h e r a t e o f r o t a t i o n a l m o t i o n o f t h e m o l e c u l e c a r r y i n g t h e chromophore. d e p o l a r i z a t i o n t o measure d i f f i c u l t y of separating  I t must be noted t h a t t h e use o f  fluorescence  can never be unambiguous because o f t h e i n t e r n a l r o t a t i o n o f t h e chromophore from t h e  r o t a t i o n o f the m o l e c u l e as a whole.  I n t h e case o f P . A . C , b o t h t h e  -83-  " p o l a r i z i n g " or a l i g n i n g of the n u c l e a r s p i n s and e m i t t i n g r a d i a t i o n come from the r a d i o a c t i v e sample whereas an i n i t i a l  external polarizing  s o u r c e i s r e q u i r e d f o r the f l u o r e s c e n c e measurements.  Gamma-rays  a r i s e from decay of e x c i t e d n u c l e a r s t a t e s r a t h e r than e x c i t e d e l e c t r o n i c s t a t e s i n the case of f l u o r e s c e n c e ( v i s i b l e r e g i o n ) . C o n s i d e r an assembly of randomly o r i e n t e d n u c l e i i n w h i c h s t a t e A decays by s u c c e s s i v e e m i s s i o n of two gamma r a d i a t i o n s , y l e v e l s B and C, as shown i n F i g u r e 6.1. isotropic.  the  t o  The t o t a l r a d i a t i o n w i l l  be  The arrangement f o r the d i r e c t i o n a l c o r r e l a t i o n experiment  i s i l l u s t r a t e d i n F i g u r e 6.2. designed  and Y2>  In the s i m p l e s t c a s e , the d e t e c t o r 1 i s  t o accept o n l y r a d i a t i o n y^ w h i c h i s e q u i v a l e n t to  choosing  n u c l e i whose magnetic d i p o l e s ( s p i n s ) a r e a l i g n e d a c c o r d i n g t o t h a t direction.  D e t e c t o r s 2 and  d e t e c t o r s may  3 are s e n s i t i v e only to r a d i a t i o n  The  count a l l photons t h a t f a l l i n t h e i r s o l i d a n g l e s ; however  the c o i n c i d e n c e a n a l y z e r s e l e c t s p r i n c i p a l l y o n l y p a i r s of r a d i a t i o n s Y^ and y^ w h i c h a r e g e n e t i c a l l y r e l a t e d t o each o t h e r .  T h i s i s accom-  p l i s h e d by a c c e p t i n g a s i g n a l from d e t e c t o r 1 o n l y i f a s i g n a l from d e t e c t o r 2 a r r i v e s a t the same t i m e .  I n o t h e r words,  which a r e e m i t t e d w i t h i n the r e s o l v i n g t i m e , T  o n l y those r a d i a t i o n s  of the c o i n c i d e n c e  circuit  K  are accepted.  T y p i c a l v a l u e s of T  a r e 10 ^ t o 10 ^ second.  By  proper  K s e l e c t i o n of r e s o l v i n g time and source s t r e n g t h , the p o s s i b i l i t y chance c o i n c i d e n c e between u n r e l a t e d r a d i a t i o n may tolerable level.  of  be reduced to a  The a n g u l a r d i s t r i b u t i o n of the second r a d i a t i o n  w i t h r e s p e c t to the f i r s t  r a d i a t i o n y^  i s expressed  Y2  as an a n g u l a r c o r r e -  l a t i o n f u n c t i o n , W(6,t) which i s a measure of the number of c o i n c i d e n c e s  -84-  A  <  \  \  \  Figure 6.1. Nuclear decay scheme of a level A which d e c a y s by the e m i s s i o n of a r a d i a t i o n tfi into a l e v e l B and t h e n by the e m i s s i o n of a r a d i a t i o n 82 into a l e v e l C .  -85-  T i counter  T j counter  Tj counter  Figure  6.2.  The  experiment, gamma-ray dipoles. (upper  and t h e o r i g i n o f  left),  that  emitted  initial  selects  nuclei  anisotropy  (i.e.,  and Y  2  in direction  y-|) at  a r e  distinguisable  unaligned nuclear magnetic  that  those rays  left).  1 8 0 ° r a t h e r than 90° to c o u n  random  to a s i n g l e  m u s t h a v e been a l i g n e d a c c o r d i n g Gamma r a y s  from those a l i g n e d n u c l e i  will  be  detectors.  subsequently observed  the d i r e c t i o n  t e r ( r i g h t ) . F o r most c a s e s ,  sufficiently different  by t h e  of  magnetic d i p o l e d i r e c t i o n s are  emission d i r e c t i o n (middle  t h e s a m p l e and t h e Y 2 " Y]  the d i r e c t i o n a l c o r r e l a t i o n  r e s t r i c t i n g the d e t e c t i o n of  preferentially  of  for  e m i s s i o n from i n i t i a l l y Although  direction to  arrangement  that  they  are  between  the  energies  readily  -86By t h i s arrangement, an a n i s o t r o p y between y^ a r r i v i n g  per u n i t time.  a t d e t e c t o r 1 and  a r r i v i n g a t d e t e c t o r s 2 and 3 i s d e t e c t e d .  If  t h e m o l e c u l e s a r e a b l e t o r o t a t e d u r i n g t h e time between t h e e m i s s i o n o f y^ and t h e observed y^, then the a n i s o t r o p y w i l l be p a r t i a l l y due  t o t h e changing n u c l e a r o r i e n t a t i o n of the nucleus  mediate s t a t e .  lost  i n the i n t e r -  F o r slow r o t a t i o n a l m o t i o n , the a n i s o t r o p y , A ( t ) w i l l  decrease e x p o n e n t i a l l y w i t h time.  Experimentally, A(t) i s obtained  from  t h e e x p r e s s i o n below  [(#Y -counts  a t l S O ^ - C Z / y ^ c o u n t s a t 90°)]  2  A ( t )  [iiy  =  2  counts a t 90°]  I n gamma-gamma s t u d i e s , f a s t e r m o l e c u l a r  (  6  ,  1  )  r o t a t i o n u s u a l l y leads t o slower  e x p o n e n t i a l decay o f a n i s o t r o p y . I n d i u m - I l l decays by e l e c t r o n c a p t u r e t o c a d m i u m - I l l  f o l l o w e d by t h e  s u c c e s s i v e e m i s s i o n o f two gamma r a y s i n t h e 173-247keV cascade as shown i n F i g u r e 6.3. F o r t h i s s o r t o f energy c a s c a d e , S t e f f e n (131)  showed  t h a t t h e c o i n c i d e n c e c o u n t i n g r a t e , W(6,t) i s g i v e n by  W(6,t) =  f-e" N  t/T  N[l+A  P (cose)]  (6.2)  where 6 i s t h e a n g l e between t h e two gamma r a y s , P 2 ( c o s 6 ) i s t h e Legendre polynomial  2 -7 l / 2 ( 3 c o s 6-1), T„(1.22x10 s e c ) i s the mean l i f e t i m e o f t h e N  i n t e r m e d i a t e n u c l e a r s t a t e f o r t h e 247keV s t a t e o f  1 : L 1  Cd  (132) , t i s t h e  t i m e i n t e r v a l between t h e e m i s s i o n o f t h e two gamma r a y s , and  *  s  a  parameter t h a t depends on t h e s p i n s and m u l t i p o l a r i t i e s a s s o c i a t e d w i t h the cascade. (131).  The t h e o r e t i c a l v a l u e f o r t h e 173-24kKeV c a s c a d e , A 2 2 0 . 1 8 =_  -87-  Eneray  Nuclear spin  420 397  7/2 "72  247  5/  (kev)  t i 2.8 days  t i = 1.2 x lcr sec ]0  173 kev,  150  11  z  =  49  min  ti - 8.5 x 10' sec z 8  2  247  0  Figure ^ C d  117  Cd  6.3. after  The  173-247  Stable  keV gamma-ray  cascade of  the e l e c t r o n - c a p t u r e decay o f  ^ I n .  -88-  F o l l o w i n g t h e e m i s s i o n o f t h e f i r s t gamma, t h e a n g u l a r  correlation  can be s t r o n g l y p e r t u r b e d by i n t e r a c t i o n o f t h e n u c l e a r q u a d r u p o l e moment 111 of  Cd i n t h e m e t a s t a b l e 111  f i e l d gradient at the  247keV s t a t e w i t h t h e f l u c t u a t i n g  electric  Cd n u c l e u s .  The p e r t u r b a t i o n on t h e a n g u l a r  c o r r e l a t i o n c a n o n l y be observed i f  i s l o n g enough and t h e n u c l e a r  moments o f t h e i n t e r m e d i a t e s t a t e a r e l a r g e enough. I n t h i s c a s e , t h e c o e f f i c i e n t o f P 2 ( c o s 6 ) i n Eq. 6.2 c a n be w r i t t e n as A 2 G 2  G22(t) i s an a t t e n t u a t i o n c o e f f i c i e n t  (133).  2 2  ( t ) where  Eq. 6.2 can then be  r e w r i t t e n as W(6,t) = j-e N  t / T  G„„(t,I,o) , n , T ) 11 o c  N[l+A G (t,I,OD ,n,T )P (cos0)] 2 2  6eQV  and  =  o  c  (6.3)  2  i s t h e p e r t u r b a t i o n f a c t o r which r e p r e s e n t s t h e i n t e r -  a c t i o n w i t h surroundings  %  2 2  h4i#-i)  »  d e t e r m i n e d by t h e parameters  n=  V  -V  -^Hzz21  ry rT parameter)  (the a8  T , where Q i s t h e quadrupole moment o f t h e i n t e r m e d i a t e s t a t e ^"''"'"Cd,  V , V , and V a r e components o f t h e f i e l d g r a d i e n t i n t h e p r i n c i p a l xx yy zz a x i s system, and T  i s the r e l a x a t i o n time.  I t i s obvious  that the  c information of p h y s i c a l i n t e r e s t i s contained perturbation factor, G  2 2  (t).  i n t h e time-dependent  I n t h e unperturbed  case,  G2 (t)=l. 2  -89-  In the p r e s e n t s t u d i e s where "'""'"''"In-porphyrin complex i s bound to a macromolecule (myoglobin) i n aqueous s o l u t i o n , the a n g u l a r i s expected  correlation  t o be p e r t u r b e d by the i n t e r a c t i o n of the n u c l e a r quadrupole  moment, Q o f '''^Cd i n the i n t e r m e d i a t e s t a t e w i t h the f l u c t u a t i n g f i e l d g r a d i e n t s a t the n u c l e u s . dynamical  The  d e t a i l s of the m o l e c u l a r  form of 0^2^)  electric  then depends on  the  motion.  The approximate form of G 2 2 ( t ) may  be d i s p l a y e d by a p l o t of the  anisotropy  =  W(TT,t)-W(7T/2,t) W(7T/2,t)  ( 1  ~ 3 "2 22 22 ' ^ A  G  (  t  a g a i n s t the d e l a y t i m e , t between t h e e m i s s i o n s . d e l a y time between the two c h a n n e l s m e n t a l l y £"22(0  r G  2 2  (r\ U  ;  The d e t a i l e d  c a n  - -2" A(t)  be determined  In p r a c t i c e  t i s the  of the c o i n c i d e n c e c i r c u i t .  Experi-  from the f o r m u l a below  W(Tr,t)-WO/2,t) W(Tr,t)+2W(ir/2,t)  shape of the p l o t of G ^ C O  ( 6  v  e  r  s  u  s  5 )  d e l a y time depends on  r e l a t i v e magnitude o f the m o l e c u l a r r o t a t i o n a l c o r r e l a t i o n t i m e , T  the  o f the  2  radioactive  probe, and upon the n u c l e a r quadrupole i n t e r a c t i o n  between the n u c l e a r q u a d r u p o l e moment (eQ) and a t the n u c l e u s  i n the i n t e r m e d i a t e  state.  (e  the e l e c t r i c f i e l d  qQ) (eq)  -90-  6.2a  Time-Dependent Quadrupole I n t e r a c t i o n - The L i m i t of Rapid M o t i o n Time-dependent quadrupole i n t e r a c t i o n s r e p r e s e n t the major p e r t u r b a t i o n  f a c t o r on a n g u l a r c o r r e l a t i o n s f o r the case i n l i q u i d s .  T h i s a r i s e s from  the Brownian m o t i o n of the m o l e c u l e s i n a l i q u i d w h i c h g i v e r i s e t o r a p i d l y f l u c t u a t i n g e l e c t r i c f i e l d g r a d i e n t s t h a t i n t e r a c t w i t h the n u c l e a r e l e c t r i c quadrupole moment, eQ i n the i n t e r m e d i a t e s t a t e . U s i n g t i m e - p e r t u r b a t i o n t h e o r y , Abragam and Pound (133) have c a l c u l a t e d the e f f e c t of r a p i d m o l e c u l a r m o t i o n on the a n g u l a r c o r r e l a t i o n  (130,134).  T h i s i s the case where m o l e c u l a r r o t a t i o n a l c o r r e l a t i o n t i m e , T  i s short c  compared t o a p e r i o d of the quadrupole f r e q u e n c y .  As an example i s the  s i t u a t i o n of a s m a l l m o l e c u l e i n a n o n - v i s c o u s l i q u i d .  For the r a p i d  m o t i o n , G ( t ) t a k e s the form of a s i m p l e e x p o n e n t i a l f o r the case of a 2 2  n u c l e a r s p i n , I = 5/2, and an a x i a l l y symmetric e l e c t r i c f i e l d  gradient  (133) G  2 2  ( t ) = exp(-A t)  (6.6)  2  where  h  - i55o<« «®\/» 2  ( 6  where double bar denotes an ensemble a v e r a g e , and T C c o r r e l a t i o n time.  -  7 )  i s the m o l e c u l a r  i s known as the r e l a x a t i o n c o n s t a n t .  The above  e q u a t i o n s assume t h a t the r o t a t i o n a l d i f f u s i o n i s i s o t r o p i c . The d i f f e r e n t i a l G  2 2  ( t ) i n Eq. 6.6 can be e x p r e s s e d i n the i n t e g r a l  form  G^F)  = 1/T jTe" N  t/T  NG (t)dt 22  (6.8)  -91-  Combining Eqs.  6.6  and  6.8  gives  G ( ~ ) = 1/(1+A T ) 22  2  The  (6.9)  n  d i f f e r e n t i a l c o r r e l a t i o n Eq.  m o n o t o n i c a l l y w i t h time. the o r i g i n a l o r i e n t a t i o n This i s also r e f l e c t e d become z e r o f o r l a r g e The  6.6  shows t h a t  G 2 2  ( t ) decreases  I n o t h e r words, a f t e r s u f f i c i e n t l y l o n g t i m e , of the assembly of n u c l e i i s c o m p l e t e l y  i n the  i n t e g r a l c o r r e l a t i o n Eq.  6.9,  lost.  which  can  f i e l d s t r e n g t h s or l o n g l i f e t i m e .  i n t e g r a l c o r r e l a t i o n i s easy to p e r f o r m e x p e r i m e n t a l l y and  much l e s s time than a measurement of d i f f e r e n t i a l G ( t ) .  However, some  2 2  i n f o r m a t i o n and  d e t a i l s c o n c e r n i n g the  in  i n t e r a c t i o n mechanism i s  usually  111 l o s t i n the a v e r a g i n g p r o c e s s .  In the p r e s e n t s t u d i e s on  m y o g l o b i n , t i m e - d i f f e r e n t i a l t e c h n i q u e was correlation  i s measured as a f u n c t i o n  From Eqs. attentuation  6.6  and  6.9,  f a c t o r v a n i s h e s and  relaxation  time, T  (Eq.  solutions:  T c  6.7)  ~10  constant, A  t h a t even f o r l a r g e  2  In other  i s o t r o p i c f o r slow motion i n l i q u i d s .  i s d i r e c t l y proportional  to the  correlation  small for small molecules i n d i l u t e  C o n s e q u e n t l y , as e v i d e n t from Eqs.  6.6  quadrupole i n t e r a c t i o n , the a t t e n t u a t i o n  the a n i s o t r o p y of a n g u l a r c o r r e l a t i o n s  and  The  6.7  of the  This explains  remains l a r g e l y u n p e r t u r b e d  s m a l l m o l e c u l e s or f r e e i o n s i n d i l u t e s o l u t i o n s .  angular why for  p r e s e n t study shows  t h a t s i m i l a r a n i s o t r o p y p a t t e r n i s observed f o r "'""'^IriMPPIX and solvents.  the  f i n i t e lower l i m i t .  c o r r e l a t i o n for small molecules i n l i q u i d s i s small.  organic  angular  of t , the d e l a y time.  t h e r e i s no  which i s u s u a l l y  "''"'"sec.  used, w h i c h means the  i t i s c l e a r t h a t f o r l a r g e v a l u e s of  words, the a n g u l a r c o r r e l a t i o n can be The  In-labelled  InTPP i n  -92-  Based on t h e Debye model (135) f o r p o l a r l i q u i d s , T  c  i s g i v e n by  where R i s t h e e f f e c t i v e m o l e c u l a r r a d i u s , £ i s t h e v i s c o s i t y , T i s the a b s o l u t e t e m p e r a t u r e , and k i s t h e Boltzman's c o n s t a n t . Eq.  6.10 c l e a r l y shows t h a t x ^ i s d i r e c t l y p r o p o r t i o n a l t o the  v i s c o s i t y of t h e l i q u i d  (135,136).  C o n s e q u e n t l y , changing t h e v i s c o s i t y  w i l l a f f e c t t h e a n i s o t r o p y of the a n g u l a r c o r r e l a t i o n s . t i m e - d i f f e r e n t i a l angular c o r r e l a t i o n s 1  Measurements of  (TDAC) on aqueous s o l u t i o n s  of  ' ' I n - C l ^ f o r v a r i o u s v i s c o s i t i e s have been performed by S t e f f e n (131). The 1  1  r e s u l t s a r e shown i n F i g u r e 6.4.  As the m o l e c u l a r r o t a t i o n d e c r e a s e s due  to increase i n s o l u t i o n v i s c o s i t y , the anisotropy decreases very  rapidly  as expected from Eq. 6.9. The dependence o f the a n i s o t r o p y on t h e v i s c o s i t y i s c l e a r l y t r a t e d by a p l o t o f G^^)  versus v i s c o s i t y  (137) i n F i g u r e 6.5.  s o l i d l i n e represents the t h e o r e t i c a l curve c a l c u l a t e d  illusThe  on t h e a s s u m p t i o n  t h a t T^ i s d i r e c t l y p r o p o r t i o n a l t o the v i s c o s i t y of t h e l i q u i d .  -93-  0  100 2O0 300 400 DELAY l ( « lc ) '  500  0  Figure  y-y  Anisotropy  6.4.  factor  ^^^-'^zZ  d i r e c t i o n a l c o r r e l a t i o n as f u n c t i o n  time t  for  ^ I n  sources of  source  is a dilute  aqueous  v i s c o s i t y was v a r i e d From S t e f f e n  (ref.  various solution  by a d d i n g 131).  of  Cd  t n e  °^  the  delay  v i s c o s i t i e s n. in  InCl^  glycerine.  whose  The  -94-  Figure 6 . 5 .  The  of  Y-Y d i r e c t i o n a l  the  the  ^ C d  viscosity  integral  n of  the  From Hemmig and S t e f f e n  anisotropy  ^ I n (ref.  factor  correlation source. 137).  G22(TO)A22  as f u n c t i o n  of  -95-  6.2b  S t a t i c E l e c t r i c F i e l d Quadrupole I n t e r a c t i o n s - P o l y c r y s t a l l i n e Samples A l t h o u g h time-dependent quadrupole  i n t e r a c t i o n s may  occur i n c r y s t a l l i n e  samples because of l a t t i c e v i b r a t i o n s and c r y s t a l d i s t o r t i o n s , s t a t i c e l e c t r i c f i e l d quadrupole  i n t e r a c t i o n i s the p r i n c i p a l p e r t u r b a t i o n f a c t o r .  Blume  (134) c o n s i d e r e d the case of a p o l y c r y s t a l l i n e sample where t h e r e i s no m o l e c u l a r r o t a t i o n a l motion a t a l l (x -*»). c G (t)  He showed t h a t G„ (t) t a k e s the  form  0  ll  = (l/5)[l+(13/7)cosoo t+(10/7)cos2a) t  2 2  o  + (5/7)cos3o) t]  (6.11)  0  (MQ = [3/21(21-1) ] (e qQ/fi)  where  2  i s the quadrupole  frequency.  The d i f f e r e n t i a l c o r r e l a t i o n i n Eq. 6.11  i s a periodic function  (a summation of c o s i n e s ) i n c o n t r a s t t o the case of time-dependent quadrupole  interaction  ( r a p i d motion) where G ( t ) decreases e x p o n e n t i a l l y . 2 2  In o t h e r words, the s t a t i c quadrupole angular c o r r e l a t i o n s .  F i g u r e 6.6  m e t a l l i c ^ " ^ I n source (138).  i n t e r a c t i o n does not d e s t r o y the  shows the measurement made w i t h a  The f a c t t h a t the e x p e r i m e n t a l p o i n t s  l i e c l o s e l y on the t h e o r e t i c a l c u r v e ( s o l i d l i n e c a l c u l a t e d t o the Eq. 6.11)  p r o v i d e s e v i d e n c e t h a t , t o a good a p p r o x i m a t i o n , o n l y  s t a t i c e l e c t r i c quadrupole indium m e t a l  according  i n t e r a c t i o n i s present i n t h i s p o l y c r y s t a l l i n e  source.  I n a p o l y c r y s t a l l i n e sample, the a n g u l a r c o r r e l a t i o n i s never wiped out c o m p l e t e l y because the s t a t i c ensemble average over a l l e m i t t i n g nuclear orientations  g i v e s a non-zero r e s u l t .  However, i n t h e case of  the time-dependent i n t e r a c t i o n s , the a n g u l a r c o r r e l a t i o n can be  completely  wiped out because the d i r e c t i o n of the f i e l d a t each n u c l e u s changes  -96-  Figure 6.6 ^ C d  y-y  Differential directional  a polycrstalline  anisotropy  correlation  In-metal  From Lehmann and M i l l e r  of  measured  source.  (ref.  A(t)  138).  the with  c o n t i n u o u s l y i n a random manner d u r i n g t h e i n t e r v a l between of  emission  and Y « 2  F i g u r e s 6.7a and 6.7b show t h e a n i s o t r o p y o f ^"^Cd gamma-gamma c o r r e l a t i o n d i s p l a y e d by p o l y c r y s t a l l i n e n o n - m e t a l l i c In^O^ and In(OH)^ (138).  The s o l i d l i n e s r e p r e s e n t  t h e t h e o r e t i c a l curves f o r  an a x i a l l y symmetric s t a t i c quadrupole i n t e r a c t i o n .  The e x p e r i m e n t a l  curves do not show t h e u s u a l c h a r a c t e r i s t i c p e r i o d i c b e h a v i o r  of the  d i f f e r e n t i a l a n i s o t r o p y A ( t ) f o r s t a t i c quadrupole i n t e r a c t i o n s ( F i g u r e s 6.7a and 6.7b) as opposed t o F i g u r e 6.6. The n o n - m e t a l l i c —8 sources  show f i r s t a r a p i d decay o f t h e c o r r e l a t i o n w i t h i n about 10 se  and t h e n p o s s i b l y f l u c t u a t e around t h e low v a l u e s .  The d i s c r e p a n c y i s  due t o t h e e f f e c t s a r i s i n g from t h e e x c i t e d e l e c t r o n s h e l l a f t e r t h e K capture  (See Appendix C ) .  -98-  IrijO,  10  15  SOURCE  20  X IO'"sec  25 t  (0)  In  (OH),  •  SOURCE  Att)  X  IO''sec  (b)  Figure 6 . 7 . ^Cd  D i f f e r e n t i a l anisotropy A ( t ) o f the  Y - Y directional correlation  observed w i t h  s o u r c e s o f (a) I i ^ O . ^ and (b) I n ( O H ) 3 > From Lehmann and M i l l e r ( r e f . 1 3 8 ) .  _gg_ 6.2c  Time-Dependent Quadrupole  I n t e r a c t i o n - The L i m i t of Slow M o t i o n  In t h e l i m i t of slow m o l e c u l a r m o t i o n , w i t h the assumption of a x i a l l y symmetric  e l e c t r i c f i e l d , M a r s h a l l and Meares (13) show t h a t the e x p r e s s i o n  for 22^ ^  i s  G  t  o b t a i n e d by m u l t i p l y i n g Eq. 6.11  by the d i f f u s i o n damping  exp(-t/xc),  factor,  3 G ( t ) = e x p ( - t / r ) l a Cos(w t ) 22 c n=o n o  (6.12)  0 0  where a  T  rot  and  J rot  = (1/6D  c  where D  a r e the c o e f f i c i e n t s i n Eq. 6.11,  n  (6.13)  i s the r o t a t i o n a l d i f f u s i o n c o n s t a n t f o r a s p h e r i c a l m o l e c u l e ,  The i n t e g r a l a t t e n t u a t i o n f a c t o r G 2 ( t )  expressed as  i s  2  G^W  =  ( l / 5 x )[1/C + (13/7) ( - 2 ^ ) o +  i n which  (5/7) (  + (10/7) C ^ q ^ > o  f ^ ) ] o  F  (6.14)  C = [(1/T„ + 1/T ) ] and N c w = (3/20)e qQ ° "ti 2  The t h e o r e t i c a l dependence of i n t e g r a l p e r t u r b a t i o n or a t t e n t u a t i o n f a c t o r 22^  °  G  n  r  o  t  a  t  i  ( s l o w and f a s t motion) log T  c  o  n  a  l  c o r r e l a t i o n time, T  c  G  cases versus  For s m a l l m o l e c u l e s i n n o n - v i s c o u s  can be as s m a l l as 10 "''"'"sec or l e s s , w h i l e f o r many p r o t e i n s , —8  T  f o r the l i m i t i n g  i s c l e a r l y i l l u s t r a t e d by a p l o t of 22^  as shown i n F i g u r e 6.8.  solutions, T  c  i s i n t h e range o f 10  sec (13).  -100Experiments by B a r r e t t by L y n d e n - B e l l (140) and  et a l . , (139)  and  theoretical  demonstrate t h a t the v a l u e of 22^ G  c o n t i n u o u s l y between those two  c  the  mechanism can no  In f a c t the environment of the  d u r i n g the n u c l e a r l i f e t i m e and  the  longer nucleus  resulting  f o r w h i c h a minimum c o r r e l a t i o n a n i s o t r o p y  e x i s t s as shown i n F i g u r e 6.8. magnitude of x^,  slow m o t i o n s ) ,  much l a r g e r than the l i f e t i m e , T„ of N  be c o n s i d e r e d as time-dependent.  i n t e r a c t i o n i s a s t a t i c one,  smoothly  6.8.  the i n t e r m e d i a t e n u c l e a r s t a t e , the p e r t u r b a t i o n  w i l l t h e n be s t a t i o n a r y  varies  l i m i t i n g cases ( r a p i d and  as shown by the b r o k e n l i n e i n F i g u r e For a c o r r e l a t i o n t i m e , x  calculations  In the r e g i o n where x^ i s of the o r d e r of  i n t e r a c t i o n i s described appropriately  neither  by  time-dependent nor by s t a t i c i n t e r a c t i o n mechanism. M a r s h a l l et a l . , (14) used parameters a s s o c i a t e d w i t h the decay of lllm^  t Q  g  e  n  e  r  a  t  e  dependence of ^22^^  t h e o r e t i c a l l y several o n  an  &^-  e  the  ( F i g u r e s 6.9-6.11).  seen from F i g u r e 6.9,  x^ can depend s t r o n g l y  illustrate  °^ attachment of the l a b e l , m o l e c u l a r  geometry o r i n t e r n a l r o t a t i o n r a t e As can be  p l o t s that  the  shape of the p l o t of G ^ C ) v e r s u s 00  on the a n g l e a t which the l a b e l i s a t t a c h e d w i t h  r e s p e c t t o the symmetry axes of the o v e r a l l m o e l c u l a r shape. F i g u r e 6.10 r o t a t i o n and  shows how  &22^°°^ v a r i e s a c c o r d i n g to the r a t e of  a n g l e of attachment of the l a b e l .  I t i s c l e a r from F i g u r e  t h a t v e r y r a p i d i n t e r n a l r o t a t i o n i n c r e a s e s the a t t e n t u a t i o n  factor,  because i t e f f e c t i v e l y reduces the magnitude of the q u a d r u p o l a r f o r a f i x e d a n g l e of attachment. i n the s i t u a t i o n where b o t h the  internal  ^22^°°^  interaction  However, i t i s i n t e r e s t i n g to n o t e i n t e r n a l r o t a t i o n of the l a b e l and  o r i e n t a t i o n o f the m o l e c u l e as a whole are  6.10  that  re-  slow, i n t e r n a l r o t a t i o n decreases  •10T-  Figure 6.8.  Theoretical  relationship between the  integral perturbation factor G (°°) and the rotational 22  correlation time T  -102-  -12  -10  -6  -8  -4  Log (T, ,| 0  Figure 6 . 9 .  Plots  of  integral  anisotropy,  G9?(»)  versus 111  rotational nucleus  correlation time,  attached  symmetry a x i s o f motion  limit  a t an a n g l e ,  (left-hand the angle of  90°>30°>0°.  In  60°>  integral  r  t  (log  e, with  set of  scale) for respect to  anisotropy  limit  varies with  90°. a l . ,  curves),  attachment,  the slow-motion  From M a r s h a l l e t  Q  a prolate molecular e l l i p s o i d .  varies with  the  T  (ref.  14)  6,  the  a  Cd  the In  the  integral  i n the order  (right-hand  main  anisotropy 60°>  set of  e i n the order  fast-  curves),  0°>30°>  -103-  03  -12  -11  -IO  -9  LOG (I/6D)  Figure 6 . 1 0 .  P l o t s of the i n t e g r a l a n i s o t r o p y  i n the  fast-motion  l i m i t versus 1og(6D), where D i s the r o t a t i o n a l d i f f u s i o n  constant  f o r a s p h e r i c a l molecule and  for  internal rotation.  Each f a m i l y of curves corresponds to a f i x e d  choice of "attachment curves (A) D^IO12,  through  1011,  1010,  i s the d i f f u s i o n constant  angle" as l i s t e d on the f i g u r e .  (D),  For  the i n t e r n a l r o t a t i o n d i f f u s i o n  individual  constant  and 10 9 s e c " 1 , r e s p e c t i v e l y . The lowest curve  i n each set (dashed l i n e ) i s the i n t e g r a l a n i s o t r o p y  i n the absence  of i n t e r n a l r o t a t i o n , or e q u i v a l e n t f o r zero "attachment From M a r s h a l l et a l . , ( r e f .  14).  angle".  -104-  L O G (6D ) M  Figure 6 . 1 1 .  P l o t s of i n t e g r a l a n i s o t r o p y versus l o g ( 6 D ^ ) , where  i s the r o t a t i o n a l d i f f u s i o n constant f o r a s p h e r i c a l macromolecule { i n the slow-motion l i m i t ) . The two s e t curves correspond to the two choices f o r the i n t e g r a l r o t a t i o n a l d i f f u s i o n constant  shown on  the f i g u r e . The i n t e g r a l a n i s o t r o p y v a r i e s w i t h the d i f f e r e n t of attachment i n the o r d e r , 0 ° > 3 0 ° > 6 0 o > 9 0 o , f o r curves a - d ,  angles  respectively.  The r e s u l t f o r e=0° i s the same as f o r no i n t e r n a l r o t a t i o n at a l l . From M a r s h a l l et a l . , ( r e f .  14).  -105-  the ^22^^  v a l u e s as i l l u s t r a t e d i n F i g u r e 6.11.  I t c a n be i n f e r r e d  from F i g u r e 6.11 t h a t when t h e m o l e c u l a r r o t a t i o n i s s l o w , i n t e r n a l r o t a t i o n does n o t a f f e c t t h e observed &22^ ^ a p p r e c i a b l y u n l e s s t h e m  r a t e o f i n t e r n a l r o t a t i o n i s s i g n i f i c a n t l y f a s t e r than t h e r a t e o f r o t a t i o n o f t h e m o l e c u l e as a whole.  I t i s i m p o r t a n t t o note  that  i n t e r n a l r o t a t i o n , whether f a s t o r s l o w , has no e f f e c t on t h e a n g u l a r c o r r e l a t i o n when t h e a n g l e o f attachment o f t h e l a b e l i s z e r o degrees. The  above d i s c u s s i o n  c l e a r l y demonstrates t h e p o t e n t i a l o f gamma-  gamma c o r r e l a t i o n t e c h n i q u e as a means t o s t u d y s p e c i f i c s i t e s on l a r g e b i o l o g i c a l l y i n t e r e s t i n g macromolecules such as hemoglobin, cytochromes. I n f o r m a t i o n l i k e t h e degree o f i n t e r n a l f l e x i b i l i t y a t t h e s i t e o f attachment o f t h e r a d i o a c t i v e  t r a c e r t o t h e macromoelcule i . e . t h e r a t e  of i n t e r n a l m o t i o n , as w e l l as t h e geometry o f t h e complex c a n be obtained. and  A l t h o u g h t h e same i n f o r m a t i o n i s a c c e s s i b l e  fluorescence depolarization,  by ESR t e c h n i q u e  t h e gamma-gamma experiment o f f e r s t h e -12  advantages o f r e q u i r i n g c o n c e n t r a t i o n s which c a n be as low as 10 and  the c a p a b i l i t y of i n vivo  experimentation.  M  -106-  6.2d  F i e l d s Without A x i a l  Symmetry  The p r e c e d i n g d i s c u s s i o n s a r e r e s t r i c t e d t o t h e cases where a x i a l l y symmetric e l e c t r i c f i e l d g r a d i e n t s (EFG) a r e assumed.  The r e s u l t s f o r  a x i a l l y symmetric EFG cases can be extended t o t h e s i t u a t i o n s o f nona x i a l l y symmetric e l e c t r i c f i e l d s by i n t r o d u c i n g an asymmetry parameter, n t o account  f o r n o n - a x i a l symmetry i n t h e quadrupole  interactions.  C o n s i d e r s t h e case o f slow m o l e c u l a r m o t i o n , Eq. 6.12 ( f o r a x i a l l y symmetric EFG) becomes  G„.(t) = e x p ( - t / T ) Ll c  3 Z b ( n ) c o s [ f (n)o> t ] n n o n=o  (6.15)  where f and t h e c o e f f i c i e n t s b (n) can be e v a l u a t e d (141). o n quadrupole  For  f r e q u e n c i e s u (n)=f (n)w a r e no l o n g e r harmonic. n n o  the  F o r n=o  ( a x i a l l y symmetric) f ^ , f ^ , and f ^ t a k e on i n t e g r a l v a l u e s . M a t t h i a s e t a l . , (142,143) have performed such c a l c u l a t i o n s on p o l y c r y s t a l l i n e sources.  F i g u r e s 6.12 and 6.13 show t h e d i f f e r e n t i a l  Q>^(t) and t i m e - i n t e g r a t e d f a c t o r , 22^° ^ ^ G  0  o r  s  P  i  n  -  v  a  l  u  e  s  I =  2  a  n  d 1=5/2  and f o r v a r i o u s v a l u e s o f t h e asymmetry parameter. 181 F i g u r e 6.14 shows t h e r e s u l t f o r t h a t w i t h p o l y c r y s t a l l i n e H^-metal s o u r c e t h e o r e t i c a l curve c a l c u l a t e d s t a t i c quadrupole  Ta gamma-gamma cascade  (144).  The s o l i d l i n e i s t h e  on t h e assumption o f an a x i a l l y  interaction.  symmetric  I t i s c l e a r l y e v i d e n t from F i g u r e 6.14  t h a t t h e r e i s a d i s c r e p a n c y between t h e e x p e r i m e n t a l and t h e o r e t i c a l results.  However, i f t h e e x p e r i m e n t a l data a r e i n t e r p r e t e d on t h e  b a s i s o f n o n - a x i a l l y symmetric quadrupole  interactions  together with  an i n c l u s i o n o f an asymmetry parameter, n=0.5 i n t o t h e c a l c u l a t i o n , an e x c e l l e n t agreement w i t h t h e e x p e r i m e n t a l r e s u l t s i s o b t a i n e d .  As  -107-  |I.2|  1.0  V-  08  0.4  0.6  G„(t)  : /<\\ /  1 \ ' ' 11\ *  0.4  /'•• \\;  1  0.2  i.j  V  \kZl/'/7''  / / •' \ *  : I T V / / «.  0  '•  ;'  1  / \; i-  \ i i  /, >\ /  to  .)  -0.2 1  .  .  .  0.5  .  1 . . .  .  i  . . . .  1.5  1.0  i . 2.0  ...<...  . 1  2.5  3.0  Ul.l/lT  • I . . . . I .... I  o3  ro  ..  Ts  I  . . . . i ....  z'o  4.5  I  3.0  0J.t/7T  Figure 6.12.  Differential  attentuation  for  rhombic quadrupole  interaction  for  intermediate  spins  is  the asymetry  From M a t t h i a s  state  parameter  142).  —  G22(t)  i n p o l y c r y s t a l l i n e sources  1=2 a n d 1 = 5 / 2 .  n=(Vxx  et a l . , (ref.  coefficients  The p a r a m e t e r  Vyy)/Vzz.  n  -108--.  Figure  6.13.  for  rhombic  for  spins  Time-integrated quadrupole  1=2  From M a t t h i a s  and et  attentuation  interaction  1=5/2. a l . ,  (ref.  143).  i  coefficients  in p o l y c r y s t a l l i n e  G (°°) 22  sources  -109=  Figure 6.14. Y-Y  Differential  directional  Hf-metal  anisotropy  A(t)  c o r r e l a t i o n measured w i t h  source.  From S c h e c t e r and S o m m e r f e l d t  (ref.  144).  of  the  Ta  a polycrystall  -110-  a m a t t e r of f a c t , t h i s was  the f i r s t i n d i c a t i o n of the presence of a  n o n - a x i a l l y symmetric s t a t i c quadrupole p e r t u r b a t i o n on a n g u l a r correlations. I t i s c l e a r from the two examples t h a t the s t r o n g dependence of a n i s o t r o p y on n (asymmetry parameter) can be used t o g i v e i n f o r m a t i o n on the geometry  of the m o l e c u l e s under s t u d y .  CHAPTER V I I  APPLICATIONS OF GAMMA-GAMMA CORRELATIONS TO BIOLOGICAL MACROMOLECULES  This chapter i s intended can be used t o o b t a i n m o t i o n a l and  t o i l l u s t r a t e b r i e f l y how P.A.C. measurements i n f o r m a t i o n on b i o l o g i c a l m a c r o m o l e c u l e s ,  f o r t h i s purpose two examples w i l l be d i s c u s s e d . Meares e t a l . , (8) a p p l i e d d i r e c t i o n a l gamma-gamma c o r r e l a t i o n s f o r  the study o f c a r b o n i c anhydrase.  The a n i s o t r o p y  o f f r e e ^^ Cd^ m  +  in  s o l u t i o n d e c r e a s e s s l i g h t l y w i t h time as i l l u s t r a t e d i n F i g u r e 7.1a. The weakly p e r t u r b e d  angular  c o r r e l a t i o n i s as expected from Eqs. 6.6  2 and  6.7 s i n c e b o t h e qQ and x ^ a r e s m a l l .  native carbonic  anhydrase, t h e a n i s o t r o p y  of t h e f r e e ^^~ Cd^ m  anisotropy  +  Even,in the presence of pattern i s s i m i l a r to that  except f o r a s l i g h t l y f a s t e r decay o f t h e gamma-ray  ( F i g u r e 7.1b).  This i s a t t r i b u t e d t o the increase i n s o l u t i o n  v i s c o s i t y by t h e a d d i t i o n o f t h e n a t i v e enzyme.  The i n c r e a s e i n s o l u t i o n  v i s c o s i t y r e s u l t s i n a slower r o t a t i o n a l d i f f u s i o n of ^ ^ C d . m  c o r r e l a t i o n i s s t r o n g l y perturbed 2+ enzyme w i t h t h e a c t i v e - s i t e Zn t h i s c a s e , x >(e^qQ) \ c  The a n g u l a r  when a p o - c a r b o n i c anhydrase (the n a t i v e removed) was added ( F i g u r e 7.1c).  In  and s i n c e t h e r o t a t i o n a l d i f f u s i o n o f t h e ^""^ Cd^ m  l a b e l l e d p r o t e i n i s now much s l o w e r ,  +  t h e r a p i d decay o f t h e a n i s o t r o p y  w i t h time i s c o n s i s t e n t w i t h Eqs. 6.6 and 6.7. The  next example c o n s i d e r s  t h e a p p l i c a t i o n o f t h e P.A.C. t e c h n i q u e f o r  i n v i v o e x p e r i m e n t a t i o n . G o o d w i n e t a l . , (145) a p p l i e d P.A.C. t e c h n i q u e t o follow ^ ^ I n  t r a c e r m e t a b o l i s m i n l i v e mice. A l t o g e t h e r  s i x indium-labelled  complexes were i n j e c t e d i n t r a v e n o u s l y i n t o i n t a c t , l i v e Swiss Webster m i c e , and  gamma-rays were counted e x t e r n a l l y .  The r e s u l t s o f t h e i n t e g r a l  -112*9  0.2  0.1  0.0  H-  -0.1  50  100  150  t (nsec)  (c) F i g u r e  7.1.  i n  v a r i o u s  i n  the  A n i s o t r o p y c h e m i c a l  p r e s e n c e  a v a i l a b l e  C d  +  +  of  3  x  - b i n d i n g  a n h y d r a s e  From  e t  a l . ,  c o r r e l a t e d  e n v i r o n m e n t s ,  a p o c a r b o n i c  Meares  of  10'^M  n a t i v e  s i t e s ) ,  (I.e.,  ( r e f .  8).  (a)  and  enzyme  gamma-ray f r e e  ^  l  c a r b o n i c  (c)  l  w i t h  l  l  m  C d  one  l  m  e m i s s i o n  C d  +  +  i n  anhydrase  +  +  i n  s t r o n g  the C d  from  ^  s o l u t i o n ; ( i . e . ,  m  C d  (b)  enzyme  p r e s e n c e  + +  1  - b i n d i n g  of  2.5  s i t e  +  i o n  +  1  1  1  m  w i t h  x  C d  +  +  no  10" 4 M  a v a i l a b l e ) .  a t t e n t u a t i o n f a c t o r , G^O") was  w  e  r  e  t a b u l a t e d i n T a b l e 7.1. When """"InCJlg  i n j e c t e d i n t r a v e n o u s l y i n t o a l i v e mice, the low G^C ) v a l u e 00  i n d i c a t e s t h a t ^ " ^ I n b i n d s t o t r a n s f e r r i n ( F i g u r e 7.2) w h i c h i s cons i s t e n t w i t h t h e o b s e r v a t i o n t h a t indium binds t o t r a n s f e r r i n q u a n t i t a t i v e l y (146,147).  almost  From Table 7.1, t h e low 22^°°^ v a l u e s f o r G  I n - c i t r a t e and In-NTA a f t e r i n j e c t i o n i n d i c a t e t h a t i n d i u m - I l l d i s s o c i a t e d from t h e complexes and bound t o t h e t r a n s f e r r i n .  The low G ^ t ) 0 0  v a l u e s r e f l e c t e d by t h e l o n g r o t a t i o n a l c o r r e l a t i o n times ( t ) a r e c h a r a c t e r i s t i c v a l u e s f o r macromolecules (8,13).  In contrast, following  the i n j e c t i o n o f In-EDTA, In-PhDTA, and In-CyDTA, t h e i n t e g r a l measurements g i v e h i g h G^^) i n t h e l i v e mice.  v a l u e s which means these compounds a r e e s s e n t i a l l y  intact  These s t u d i e s by Goodwin e t a l . , (145) c l e a r l y demonstrate  the p o t e n t i a l of the  P.A.C. t e c h n i q u e i n d e t e r m i n i n g t h e f a t e o f  "'""'""'"In-labelled t r a c e r s i n humans by t h e e x t e r n a l c o u n t i n g s o f t h e gammar a y s e m i t t e d by t h e r a d i o a c t i v e t r a c e r . There remains a few remarks c o n c e r n i n g t h e s o r t o f i n f o r m a t i o n t h a t can be e x t r a c t e d from t h e shape o f t h e p l o t o f a n i s o t r o p y v e r s u s d e l a y time.  As mentioned i n S e c t i o n 6.2c,  l a b e l s can be determined spectrum. molecules  t h e a n g l e o f attachment o f t h e  by a n a l y z i n g t h e t i m e - d i f f e r e n t i a l c o i n c i d e n c e  I f t h e spectrum shows a s e r i e s o f minima, t h e l a b e l l e d a r e g e n e r a l l y o r i e n t e d i n t h e same d i r e c t i o n .  between the minima w i l l be determined  The s p a c i n g  by t h e o r i e n t a t i o n o f t h e p r i n c i p a l  a x i s o f an o r d e r e d a r r a y o f m o l e c u l e s w i t h r e s p e c t t o t h e d i r e c t i o n o f t h e f i r s t gamma e m i s s i o n . ordered arrays of molecules  Tendon, f i b e r s , and membranes a r e examples o f t h a t may be s t u d i e d by t h e P.A.C. method.  s t u d y o f c o n f o r m a t i o n a l changes i n p o l y p e p t i d e c h a i n s ( e s p e c i a l l y  The  helix-  random c o i l t r a n s i t i o n s ) h a s been a s u b j e c t o f c o n s i d e r a b l e i n t e r e s t . A  Table 7.1.  Compound  In Compounds S t u d i e d In Vivo  *PAC  Column Chromatography (%) Transferrin Compound  RIEt Transferrin  No. o f Mice  Anisotropy=}=  In-transferrin  7  0.076±0.004  100  0  Positive  In-citrate  4  0.081±0.004  100  0  Positive  In-NTA  3  0.080±0.005  100  0  Positive  In-o-PhDTA  3  0.122±0.007  9.8  90.2  Negative  In-CyDTA  3  0.128±0.005  10.7  89.3  Negative  In-EDTA  3  0.120±0.007  10.2  89.8  Negative  * PAC = P e r t u r b e d Angular t RIE =  Correlation  Radioimmunoelectrophoresis  =j= Mean o f A±2 S.D. From Goodwin e t a l . , ( r e f .  145).  Band  -115-  Figure  7.2.  intravenous Initial  In  vivo  i n j e c t i o n of  low v a l u e s  In-EDTA i s n o t From G o o d w i n  angular  et  for  protein a l . ,  c o r r e l a t i o n s of  three  citrate  l a b e l l e d i n d i u m compounds  following into  mice.  and c h l o r i d e i n d i c a t e p r o t e i n - b i n d i n g .  bound.  (ref.  gamma-rays  145).  -116f a i r l y r i g i d h e l i c a l conformation trum w i t h  a s i n g l e minimum.  i s expected t o g i v e a c o i n c i d e n c e  spec-  However, upon t r a n s i t i o n t o a random c o i l  e f f e c t e d by pH change, t h e i n c r e a s e i n f l e x i b i l i t y o f t h e c h a i n i s r e f l e c t e d by an e x p o n e n t i a l decay curve c h a r a c t e r i s t i c o f f r e e molecules i n solution.  T h i s i s i l l u s t r a t e d i n F i g u r e 7.3 i n w h i c h M a r t i n  et a l . , (148) s t u d i e d t h e h e l i x - r a n d o m a c i d by P.A.C.  c o i l t r a n s i t i o n i n glutamic  S i n c e P.A.C. i s a p p l i c a b l e t o s o l i d s , i t means s u r f a c e  l a y e r s , powders, composite m a t e r i a l s e t c . , can a l s o be s t u d i e d . P.A.C. can a l s o be used t o d e t e r m i n e t h e s i z e o f a c l e f t i n a macromolecule by a t t a c h i n g an a p p r o p r i a t e r o t a t i o n a l t r a c e r o f known s i z e t o the c l e f t .  I f a small r o t a t i o n a l tracer i s incorporated  i n t o t h e c l e f t , the g r e a t e r movement o f the t r a c e r i s r e f l e c t e d by an e x p o n e n t i a l decay i n t h e t i m e - d i f f e r e n t i a l c o i n c i d e n c e spectrum.  On  the o t h e r hand, i f a s i n g l e minimum o c c u r s , the r o t a t i o n a l t r a c e r i s r i g i d l y attached  t o t h e m o l e c u l e and moves w i t h a r o t a t i o n a l  time t h a t i s l o n g compared t o t h e quadrupolar  interaction.  correlation  10  20 CHANNEL  Figure 7.3.  Time-differential  30 NUMBER  perturbed angular c o r r e l a t i o n (TDPAC)  s p e c t r a f o r samples of p o l y g l u t a m i c a c i d at pH=4.0 (upper curve) and pH=7.8 (lower c u r v e ) .  The upper curve corresponds to the t i g h t  conformation whereas the lower curve i s f o r the random c o i l  helix  configuration.  -118-  CHAPTER V I I I EXPERIMENTAL WORK  8.1  P r e p a r a t i o n of M e s o - p r o t o p o r p h y r i n Meso-protoporphyrin  IX  I X (MPP IX) was chosen f o r t h e p r e s e n t  study.  T h i s p o r p h y r i n i s more s t a b l e than p r o t o p o r p h y r i n I X because t h e v i n y l groups o f t h e p r o t o p o r p h y r i n IX a r e c h e m i c a l l y and p h o t o c h e m i c a l l y labile.  As an example, when a s o l u t i o n o f p r o t o p o r p h y r i n I X i n benzene  o r p y r i d i n e i s exposed t o l i g h t , an o x i d a t i o n product p r o t o p h y r i n I X i s formed (149).  known as photo-  However, h y d r o g e n a t i o n  of t h e v i n y l  groups i s accompanied by an u n d e s i r a b l e r e d u c t i o n i n aqueous s o l u b i l i t y o f the r e s u l t i n g m e s o - p r o t o p o r p h y r i n  IX.  Materials: F e r r i - p r o t o p o r p h y r i n IX was o b t a i n e d from Strem C h e m i c a l s , I n c . ; palladium oxide  (PdO. x ^ 0 ) was from MCB; f o r m i c a c i d  (90%) was o b t a i n e d  from F i s h e r ; ammonia from A l l i e d Chemical Canada, L t d . ; ammonium a c e t a t e from MCB; sodium t a r t r a t e AR was o b t a i n e d from M a l l i n c k r o d t .  Procedure: The p r o c e d u r e f o r t h e p r e p a r a t i o n of m e s o - p r o t o p o r p h y r i n f e r r i - p r o t o p o r p h y r i n IX was e s s e n t i a l l y t h a t of T a y l o r (150). method i n v o l v e d t h e s i m u l t a n e o u s  I X from The  reduction of the v i n y l sidechains to  e t h y l groups and removal o f i r o n through  t h e a c t i o n of f o r m i c a c i d .  The p a l l a d i u m o x i d e was used as t h e r e d u c i n g c a t a l y s t .  Ferro-proto-  p o r p h y r i n IX was i n i t i a l l y formed by t h e r e d u c t i o n o f F e ( I I I ) t o F e ( I I ) . o  S i n c e F e ( I I ) has a l a r g e r i o n i c r a d i u s , 0.74A compared w i t h t h a t o f o  Fe(III),  0.64A, F e ( I I ) was more r e a d i l y d i s s o c i a t e d from t h e p o r p h y r i n  -119-  m a c r o c y c l e , thus p r o t o p o r p h y r i n IX was o b t a i n e d .  The f i n a l  product  m e s o - p r o t o p o r p h y r i n IX was o b t a i n e d by c a t a l y t i c h y d r o g e n a t i o n of the v i n y l groups of p r o t o p o r p h y r i n IX. F e r r i - p r o t o p o r p h y r i n IX (2.0g) was  suspended  (90%; 165 ml) c o n t a i n i n g p a l l a d i u m o x i d e (460 mg) bottom f l a s k f i t t e d w i t h a r e f l u x condenser. p r o t e c t e d from l i g h t by aluminium f o i l .  i n formic acid  i n a 250 ml  The whole arrangement was  The m i x t u r e was  1% h r , and the p r o g r e s s of the r e a c t i o n was  round-  refluxed for  followed spectroscopically.  T h i s was a c h i e v e d by w i t h d r a w i n g an a l i q u o t of the r e a c t i o n m i x t u r e , and then d i l u t i n g w i t h f o r m i c a c i d . was  The v i s i b l e a b s o r p t i o n spectrum  then compared w i t h t h a t o f p r o t o p o r p h y r i n IX ( F i g u r e s 8.1 and  8.2).  C o m p l e t i o n of t h e r e a c t i o n was determined by the d i s a p p e a r a n c e o f the p r o t o p h y r i n IX spectrum.  The p a l l a d i u m o x i d e was  f i l t e r e d o f f using a  s i n t e r e d g l a s s f u n n e l o f f i n e p o r o s i t y , and was washed w i t h f o r m i c a c i d The f i l t r a t e was  then poured  with e f f i c i e n t s t i r r i n g . p r e c i p i t a t e was  i n t o 30% ammonium a c e t a t e s o l u t i o n (600 ml)  A f t e r b e i n g a l l o w e d t o stand f o r 45 min,  c o l l e c t e d by c e n t r i f u g a t i o n and  deionised water.  The p r e c i p i t a t e was  washed r e p e a t e d l y w i t h  taken up i n 2% aqueous ammonia  s o l u t i o n (140 ml) to g i v e a b r o w n i s h - r e d s o l u t i o n . m e s o - p r o t o p o r p h y r i n IX was s o l u t i o n (24 m l ) .  The sodium s a l t of  " s a l t e d o u t " w i t h 30% sodium  The p r e c i p i t a t e was  the  tartrate  c o l l e c t e d by c e n t r i f u g a t i o n , and  i f the s u p e r n a t a n t remained b r o w n i s h - r e d , the above procedure  was  repeated. The sodium s a l t  was  c o n v e r t e d i n t o m e s o - p r o t o p o r p h y r i n IX hydro-  c h l o r i d e by the a d d i t i o n of b o i l i n g 25% h y d r o c h l o r i c a c i d (120 ml) t o the p r e c i p i t a t e i n the c e n t r i f u g e tubes.  The s o l i d d i s s o l v e d  and c r y s t a l s o f the h y d r o c h l o r i d e formed almost i m m e d i a t e l y .  instantly The  -120-  c r y s t a l l i n e m e s o - p r o t o p o r p h y r i n IX h y d r o c h l o r i d e was  c o l l e c t e d by  f i l t r a t i o n , washed w i t h a l i t t l e 2.5%  hydrochloric acid solution,  and d r i e d i n a i r .  achieved  F u r t h e r d r y i n g was  t a i n i n g p e l l e t s of sodium  hydroxide.  i n a d e s i c c a t o r con-  - 1 2 1 -  _ J  349  I  U2U  I  I  Wavelength  F i g u r e f o r m i c  8.1. a c i d .  V i s i b l e  I  500  a b s o r p t i o n  551  I  L  625  700  nm  spectrum  of  p r o t o p o r p h y r i n  IX  i n  -122-  I  1  I  383  460  473  I  306  WAVELENGTH F i g u r e 8.2.  I  548  I  624  L  700  nm  V i s i b l e a b s o r p t i o n spectrum of meso-protoporphyrin  in formic a c i d .  IX  -123-  8.2  P r e p a r a t i o n o f Non-Radioactive Indium-Mesoprotoporphyrin IX  Materials: Indium c h l o r i d e , . I n C l ^ (anhydrous, u l t r a - p u r e ) was o b t a i n e d from Alfa;  reagent g l a c i a l a c e t i c a c i d from A l l i e d Chemical Canada, L t d . ;  s i l i c a g e l (70-140 mesh) f o r column chromatography  was o b t a i n e d from  Macherey, N a g e l & Co., Germany; and m e s o - p r o t o p o r p h y r i n I X was p r e p a r e d i n t h e l a b o r a t o r y a c c o r d i n g t o t h e p r o c e d u r e i n S e c t i o n 8.1. Procedure: In a 250 ml one-necked  round-bottom  flask f i t t e d with a reflux  condenser, i n d i u m c h l o r i d e (0.148g) and m e s o - p r o t o p o r p h y r i n I X (0.200g) were heated t o b o i l i n g i n g l a c i a l a c e t i c a c i d (160 ml) c o n t a i n i n g a c e t a t e (0.717g). foil.  sodium  The r e a c t i o n was p r o t e c t e d from l i g h t by aluminium  R e f l u x i n g was c o n t i n u e d f o r 4% h r .  Completion o f t h e m e t a l l a t i o n  p r o c e s s was determined s p e c t r o s c o p i c a l l y by t h e d i s a p p e a r a n c e o f t h e v i s i b l e spectrum o f m e s o - p r o t o p o r p h y r i n I X . The s o l u t i o n was conc e n t r a t e d on a r o t a r y e v a p o r a t o r t o a m i n i m a l volume.  A d d i t i o n of  water t o t h e warm, c o n c e n t r a t e d s o l u t i o n produced a c o p i o u s p r e c i p i t a t e of b r i g h t - r e d i n d i u m - m e s o p r o t o p o r p h y r i n IX (InMPP I X ) .  The s o l i d was  c o l l e c t e d , washed w i t h water and d r i e d over p e l l e t s o f sodium h y d r o x i d e i n vacuo.  -124-  8.2a  T h i n - l a y e r chromatography A n a l y t i c a l t h i n - l a y e r chromatography  p u r i t y of InMPP IX. fluorescent  ( t i c ) was  used to check the  Eastman chromatogram sheets of s i l i c a g e l w i t h  i n d i c a t o r were used.  A f t e r an e x t e n s i v e s e a r c h , the best  s o l v e n t system f o r the development of the chromatograms was of toluene/methanol (2:1).  No  s p r a y i n g agent was  a  mixture  r e q u i r e d to see  spots s i n c e components of i n t e r e s t were i n t e n s e l y c o l o r e d .  Figure  the 8.3  shows the t i c r e s u l t s w i t h v a r i o u s s o l v e n t systems.  ORIGIN A  B T:M(1:1)  F i g u r e 8.3.  T:M(2:1)  DH(5--1)  T:M(10--2)  TLC of (A) meso-tetraphenylporphine IX and (B) indium meso-tetraphenylporphyrin IX A b b r e v i a t i o n s : s o l v e n t s : T, t o l u e n e ; M, methanol; H, water; L, 2 , 6 - L u t i d i n e .  -125-  8.2b  P u r i f i c a t i o n of I n d i u m - M e s o p r o t o p o r p h y r i n IX From the t i c r e s u l t s ( S e c t i o n 8.2a), t h e crude InMPP IX  (100 mg) was d i s s o l v e d i n m i n i m a l methanol and chromatographed 62 cm x 1.6  cm column o f s i l i c a g e l packed i n t o l u e n e .  e l u t e d w i t h toluene/methanol (2:1). by u n r e a c t e d MPPIX. by t i c . 8.2c  InMPP IX was  complex on a  The column was  eluted f i r s t  followed  A l l f r a c t i o n s were checked s p e c t r o s c o p i c a l l y and  The s o l v e n t was removed i n vacuo t o r e c o v e r t h e b r i g h t - r e d  InMPPIX.  V i s i b l e A b s o r p t i o n Spectroscopy The v i s i b l e a b s o r p t i o n s p e c t r a o f i n d i u m - m e s o p r o t o p o r p h y r i n IX  i n f o r m i c a c i d and benzene were r e c o r d e d on a Cary Model 14 photometer.  spectro-  On f o r m a t i o n of InMPP, the i n t e n s i t y of t h e ct-band  i n c r e a s e d s i g n i f i c a n t l y t o almost t h a t of the 8-band (see F i g u r e s 8.4 and 8.5). A  The two bands were h y p o c h r o m i c a l l y s h i f t e d , the ot-band has  a t 573 nm and t h e 8-band a t 560  nm.  -126-  399  536 573  I  I  335 F i g u r e 8.4.  L _ l  I  I  460 UU 548 Wavelength nm  I  625  V i s i b l e a b s o r p t i o n spectrum of indium meso-protoporphyrin  i n formic a c i d .  IX  -127-  F i g u r e 8.5. i n benzene.  V i s i b l e a b s o r p t i o n spectrum of indium meso-protoporphyrin  IX  -128-  8.3  Preparation of Radioactive I n d i u m - I l l Meso-protoporphyrin  IX  The normal method o f p r e p a r i n g indium-MPP i s one o f r e f l u x i n g l a r g e excess o f i n d i u m and t h e p o r p h y r i n as d i s c u s s e d i n S e c t i o n 8.2. T h i s method i s n o t a p p l i c a b l e t o t h e s y n t h e s i s o f r a d i o a c t i v e i n d i u m - I l l MPPIX s i n c e t h e molar r a t i o o f r a d i o a c t i v e i n d i u m - I l l t o MPPIX i s e x t r e m e l y low.  T h e r e f o r e a s l i g h t l y d i f f e r e n t method was developed.  The s e a r c h f o r t h e optimum r e a c t i o n c o n d i t i o n s was c a r r i e d out w i t h " c o l d " i n d i u m i n v i e w o f t h e f a c t t h a t i n d i u m - I l l emits gamma r a y s . S e v e r a l f a c t o r s were c o n s i d e r e d : r e f l u x i n g t i m e , amount o f " c o l d " i n d i u m p r e s e n t was c r i t i c a l s i n c e b o t h " h o t " and " c o l d " indium would compete f o r t h e p o r p h y r i n , and t h e amount o f MPP.  The method d e s c r i b e d  here was o b t a i n e d a f t e r an e x t e n s i v e s e a r c h f o l l o w i n g sometimes d i s a p p o i n t i n g r e s u l t s o f low r a d i o a c t i v i t y o f the r e c o n s t i t u t e d m y o g l o b i n with ^^InMPPIX.  A h i g h r a d i o a c t i v i t y o f ^''"InMPPIX was e s s e n t i a l t o  t h e s u c c e s s o f t h e subsequent gamma-gamma experiments  on t h e r e c o n s t i t u t e d  myoglobin. A l l t h e experiments  were c a r r i e d out w i t h i n an i m p r o v i z e d l e a d  (3 mm t h i c k ) e n c l o s u r e o f 50 cm x 50 cm x 40 cm i n a fume-hood.  This  p l a c e d a s e v e r e l i m i t a t i o n on t h e v i s i b i l i t y and c h e m i c a l manipul a t i o n s . S i n c e t h e h a l f - l i f e o f i n d ium-111 i s s h o r t (tj=2.8 d a y s ) , a l l g l a s s w a r e s and r a d i o a c t i v e contaminated  s o l u t i o n s and a r t i c l e s were  s t o r e d i n t h e l e a d e n c l o s u r e u n t i l t h e r a d i o a c t i v i t y was no l o n g e r detectable before disposal. for  A P i c k e r Model 642081 Labmonitor was used  d e t e r m i n i n g r a d i a t i o n l e v e l s , e s t i m a t i n g sample a c t i v i t y , and c h e c k i n g  contamination. Materials: In-111 was o b t a i n e d c o m m e r c i a l l y as c a r r i e r - f r e e •'•^^InClo i n  -139aqueous s o l u t i o n c o n t a i n i n g  0.45%  t o 0.9%  sodium c h l o r i d e from Medi-  P h y s i c s Corp., E m e r y v i l l e , C a l i f o r n i a . Procedure: The method was e s s e n t i a l l y s i m i l a r t o t h a t d e s c r i b e d i n S e c t i o n 8.2. IX  I n a 25 ml round-bottom f l a s k f i t t e d w i t h a r e f l u x condenser,  (6 mg),  "cold" I n C l  g l a c i a l acetic acid  3  (2.3 mg), sodium a c e t a t e (9.1 mg) i n  (6 ml) was s t i r r e d v i g o r o u s l y .  ^"^InCl^  solution  (1.1 m l ; 2.2 mCi) was i n j e c t e d i n t o the r e a c t i o n m i x t u r e w h i c h refluxed  f o r 6-7 h r .  Addition  of c o l d d e i o n i z e d  c o n c e n t r a t e d s o l u t i o n produced a b r i g h t - r e d  to p u r i f y the p r o d u c t .  w a t e r t o the warm,  p r e c i p i t a t e which  washed r e p e a t e d l y w i t h c o l d water and a i r - d r i e d .  the Labmonitor.  was  The s o l u t i o n was then c o n c e n t r a t e d t o a m i n i m a l  volume by e v a p o r a t i o n .  with  MPP  was  No attempt was made  The r a d i o a c t i v i t y of the p r o d u c t was  checked  The "^"'"InMPPIX was then i m m e d i a t e l y used f o r the  r e c o n s t i t u t i o n e x p e r i m e n t s (See S e c t i o n  8.5).  •-1308.4  P r e p a r a t i o n o f Apomyoglobin S o l u t i o n  Materials: M y o g l o b i n from spermwhale s k e l e t a l muscle type I I ( s a l t - f r e e l y o p h i l l i s e d powder) was o b t a i n e d from Sigma. e t h y l ketone) was o b t a i n e d  2-Butanone  (methyl  from F i s h e r .  Procedure: The  procedure was s i m i l a r t o t h a t r e p o r t e d by B r e s l o w (151).  A  500 mg sample o f m y o g l o b i n was d i s s o l v e d i n 60 ml d e i o n i z e d water (VL% s o l u t i o n ) a t 0°C, and t h e pH c a r e f u l l y a d j u s t e d t o 1.5 w i t h IN HC£. The removal o f t h e heme from myoglobin was i n s u r e d i f t h e pH o f t h e i n i t i a l met-Hb s o l u t i o n was near 1.5 w h i c h i s a c r i t i c a l for  the cleavage  o f t h e protein-heme l i n k .  value  The s o l u t i o n was then  e x t r a c t e d a t 4°C f i r s t w i t h an e q u a l volume o f 2-butanone, then t w i c e more w i t h a h a l f - v o l u m e  o f 2-butanone t o remove t h e heme.  The l a s t  e x t r a c t i o n was g e n e r a l l y found t o be s u p e r f l u o u s . The r e s u l t i n g , s t r a w - c o l o r e d s o l u t i o n was then immediately changes o f sodium b i c a r b o n a t e  slightly  d i a l y z e d a t 4°C a g a i n s t 4  s o l u t i o n (50 mg/l l i t r e ) , and t h e n a g a i n s t  s e v e r a l changes o f d e i o n i z e d w a t e r .  The apomyoglobin s o l u t i o n was -4  f u r t h e r d i a l y z e d a g a i n s t 2 changes o f 1 x 10  M disodium  and a g a i n s t s e v e r a l changes o f d e i o n i z e d w a t e r . was  EDTA s o l u t i o n ,  The apomyoglobin s o l u t i o n  c e n t r i f u g e d t o remove d e n a t u r e d p r o t e i n w h i c h had p r e c i p i t a t e d , and was  concentrated  t o M.0 ml by u l t r a - f i l t r a t i o n u s i n g an Amicron arrangement  f i t t e d w i t h a UM-2 membrane (>1000 M.W.).  F i n a l l y , the concentrated  p r o t e i n s o l u t i o n was d i a l y z e d a g a i n s t 4 changes o f 250 ml sodium b o r a t e b u f f e r (pH 9.2; i o n i c s t r e n g t h 0.16). was  The c o n c e n t r a t i o n o f apomyoglobin  determined based upon i t s absorbance a t 280 nm (e = 15,500).  The  apomyoglobin s o l u t i o n was i m m e d i a t e l y used f o r r e c o n s t i t u t i o n w i t h 11:L  I n M P P I X (See S e c t i o n 8.5).  -1328.5  R e c o n s t i t u t i o n o f Apomyoglobin w i t h I n d i u m - I l l M e s o - p r o t o p o r p h y r i n IX A l l t h e r e c o n s t i t u t i o n experiments were c a r r i e d o u t w i t h i n t h e l e a d  e n c l o s u r e i n a fume-hood as d i s c u s s e d i n S e c t i o n 8.3. As mentioned i n S e c t i o n 8.3, t h e l i m i t e d v i s i b i l i t y  (due t o t h e l e a d e n c l o s u r e ) had  been a s e v e r e h a n d i c a p , hence a l e a d g l a s s s h i e l f (32 cm x 24 cm)  was  used d u r i n g column chromatography. Materials: Sephadex G-25 was o b t a i n e d from Pharmacia F i n e C h e m i c a l s , Sweden; carboxy m e t h y l c e l l u l o s e , CM-52 ( m i c r o g r a n u l a r , p r e s w o l l e n ) , a c a t i o n exchanger f o r column chromatography was o b t a i n e d from Whatman, England. ^"^InMPPIX was o b t a i n e d from t h e method d i s c u s s e d i n S e c t i o n 8.3.  Apo-  m y o g l o b i n was p r e p a r e d as d e s c r i b e d i n S e c t i o n 8.4. Procedure: The p r o c e d u r e of r e c o n s t i t u t i o n was t h a t o f S r i v a s t a v a  (152).  "'""'""''InMPPIX was d i s s o l v e d i n a m i n i m a l volume o f 0.1N NaOH and i m m e d i a t e l y d i l u t e d t e n - f o l d w i t h b o r a t e b u f f e r (pH=9.2; i o n i c s t r e n g t h = 0.16). The ^''"InMPPIX s o l u t i o n was i m m e d i a t e l y added t o 4 ml o f apomyoglobin s o l u t i o n -4 [1.6 x 10  H/l]  a t 0°C w i t h s t i r r i n g .  The r e s u l t i n g s o l u t i o n was then  s t o r e d a t 0°C i n a Dewar f l a s k f o r about 8 h r . i m p r a c t i c a l i n v i e w o f t h e h i g h gamma e m i s s i o n s .  Normal r e f r i g e r a t i o n was The s o l u t i o n was then  chromatographed over Sephadex G-25 (48 cm x 1 cm column) w h i c h had been e q u i l i b r a t e d w i t h 0.005M phosphate b u f f e r , pH6.3 and e l u t e d w i t h t h e same b u f f e r .  T h i s s t e p removed excess u n r e a c t e d "''"''"'"InMPPIX,  A  r e d d i s h band c o r r e s p o n d i n g t o t h e r e c o n s t i t u t e d m y o g l o b i n was e l u t e d w i t h i n t h e v o i d volume o f t h e column f o l l o w e d by t h e u n r e a c t e d "'^InMPPIX. To remove "'""'""'"InMPPIX t h a t were bound t o n o n - a c t i v e s i t e s on t h e p r o t e i n s ,  -133-  the e l u t e d p r o t e i n was column (8 cm x 2 cm)  absorbed on a CM-52 c e l l u l o s e c a t i o n exchange  e q u i l i b r a t e d w i t h 0.005M phosphate, pH 6.3  (below  the i s o e l e c t r i c p o i n t , s i n c e the p l v a l u e of m y o g l o b i n i s 7.0). CM-column was  constructed  shown i n F i g u r e 8.6. w h i l e the  The  from a 30 cc p l a s t i c d i s p o s a b l e  The  syringe  as  p r o t e i n absorbed s t r o n g l y on the c e l l u l o s e  InMPPIX passed s t r a i g h t t h r o u g h .  u n t i l no ^""^InMPPIX ( r e d d i s h c o l o r ) was  E l u t i o n was  collected.  The  continued  eluent  was  f i n a l l y changed to 0.05M phosphate, pH7.0, the p r o t e i n (a deep red band) i m m e d i a t e l y moved down the column and was a c t i v i t y of the r e c o n s t i t u t e d sample was Labmonitor.  collected.  e s t i m a t e d w i t h the  The  radio-  Picker  -134-  rubberseptum syringe neddle  syringe  neddle  rubber 30cc  cork  plastic  disposable  syringe  CM-52 gel  sand glass  wool Tygon  Figure  tubing  8.6. A s c h e m a t i c d r a w i n g o f a CM-52 c o l u m n  constructed  from a 30cc p l a s t i c d i s p o s a b l e  syringe.  -135-  8.6  P e r t u r b e d A n g u l a r C o r r e l a t i o n Measurements The e x p e r i m e n t a l t e c h n i q u e i s concerned  w i t h t h e measurement o f  the a n g u l a r d i s t r i b u t i o n o f t h e second gamma e m i s s i o n w i t h r e s p e c t t o the d i r e c t i o n o f t h e f i r s t gamma e m i s s i o n .  This r e q u i r e s detectors  and e l e c t r o n i c equipment w h i c h w i l l be d e s c r i b e d b r i e f l y below. 8.6a  Detectors S c i n t i l l a t i o n d e t e c t o r s i n s t e a d o f G e i g e r c o u n t e r s were used because o f  t h e i r h i g h e f f i c i e n c y and good energy r e s o l u t i o n .  They a l s o p r o v i d e  f a s t e r p u l s e s w i t h much l e s s u n c e r t a i n t y i n t i m e than G e i g e r c o u n t e r s w h i c h i s e s s e n t i a l i n measurements r e q u i r i n g c o i n c i d e n c e d e t e c t i o n . T h a l l i u m - a c t i v a t e d sodium i o d i d e d e t e c t o r s were used f o r t h e d e t e c t i o n o f gamma e m i s s i o n s .  The h i g h Z o f t h e i o d i n e p r o v i d e s a  l a r g e p h o t o e l e c t r i c c r o s s - s e c t i o n which a l l o w s one t o choose t h e gammar a y d e s i r e d by p u l s e - h e i g h t s e l e c t i o n .  The r e l a t i v e l y l o n g phosphor  decay time o f 0.25 ysec may appear t o be t o o l o n g f o r f a s t c o i n c i d e n c e work; however, by o p e r a t i n g a f a s t c o i n c i d e n c e c i r c u i t on t h e i n i t i a l p a r t o f t h e p u l s e , r e s o l v i n g times o f o n e - t e n t h  t o one-hundred o f t h e  decay time c o u l d be a c h i e v e d f o r gamma r a y s o f 100 keV o r more. P h o t o m u l t i p l i e r tubes were used t o d e t e c t p u l s e s from t h e d e t e c t o r s , and a t t h e same time t o p r o v i d e an a m p l i f i c a t i o n f a c t o r up t o 10 . The a m p l i f i e d e l e c t r i c a l p u l s e s were s u f f i c i e n t l y l a r g e t o o p e r a t e t h e e l e c t r o n i c d e t e c t i o n system.  The p h o t o m u l t i p l i e r s were p r o t e c t e d from  h i g h magnetic f i e l d w i t h magnetic s h i e l d i n g .  -136-  8.6b  Electronics A s i m p l i f i e d schematic  diagram o f the f a s t - s l o w c o i n c i d e n c e  c i r c u i t i s shown i n F i g u r e 8.7.  R a d i a t i o n s f a l l i n g w i t h i n the s o l i d  a n g l e s o f t h e d e t e c t o r s were counted.  However, t h e c o i n c i d e n c e  a n a l y z e r was used t o s e l e c t p r i n c i p a l l y o n l y p a i r s o f r a d i a t i o n s and  w h i c h a r e g e n e t i c a l l y r e l a t e d t o each o t h e r ( i . e . e m i t t e d  i n the same n u c l e a r d e c a y ) .  The e l e c t r i c a l p u l s e s from the d e t e c t o r s  were c h a n e l l e d i n t o two l i n e s , " f a s t " and "slow" of 10  -6  s e c , f a s t was 10  -7  t o 10  -9  (slow was i n t h e o r d e r  sec p u l s e w i d t h ) .  A fast  coincidence  c i r c u i t was e s s e n t i a l t o d i s c r i m i n a t e a g a i n s t chance c o i n c i d e n c e s a t high counting rates. fast coincidence  P u l s e s on t h e f a s t l i n e s were a p p l i e d to the  circuit.  The p u l s e s were a t the same time a p p l i e d i n sequence t o slow l i n e a r a m p l i f i e r s and t o p u l s e - h e i g h t a n a l y z e r s f o r energy s e l e c t i o n . The p u l s e h e i g h t was p r o p o r t i o n a l t o the energy o f the i n c i d e n t photon. The o u t p u t s o f the a n a l y z e r s t o g e t h e r w i t h the output of t h e f a s t c o i n c i d e n c e c i r c u i t were then a p p l i e d t o a slow m u l t i p l e c o i n c i d e n c e c i r c u i t t h a t s e l e c t s the event. m u l t i - c h a n n e l analyzer f o r data  The output p u l s e was then passed t o a storage.  I n the p r e s e n t s t u d y , the a n g u l a r d i s t r i b u t i o n was s t u d i e d as a f u n c t i o n o f t h e time between the f o r m a t i o n o f t h e o r i e n t e d s t a t e and i t s decay, t h a t i s t h e time between the a r r i v a l o f the two gamma r a y s . T h i s was a c h i e v e d by i n t r o d u c i n g an e l e c t r o n i c d e l a y i n t h e l i n e o f t h e f i r s t detector.  •137-  Smgie Channel Amplif ie  Pulse Height  r  Analyzer | —\  Post Amplifier  Post  Slow  Coincidence  Coincidence  Output  Cucutl  Circuit  Fast Amplifie-  -J  Slow  Single  Chonnel  Pulse  Height  Amplifier  Figure 8.7. arrangement  Analyier  A schematic diagram f o r for  P.A.C.  From F r a u e n f e l d e r  a fast-slow coincidence  measurements.  and S t e f f e n  (ref.  130).  -138H  8.6c  Method A t h r e e - d e t e c t o r f a s t - s l o w gamma r a y c o i n c i d e n c e  was used.  The d e t e c t o r s were arranged  r a d i o a c t i v e sample.  spectrometer  a t c a r d i n a l p o i n t s around t h e  The P.A.C. measurements a t 180° and 90° were  performed s i m u l t a n e o u s l y u s i n g t h r e e 2" x 2" d i a . NaI(T£) d e t e c t o r s . A s i m p l i f i e d diagram o f the f a s t - s l o w c o i n c i d e n c e c i r c u i t used f o r t h e p r e s e n t measurements i s shown i n F i g u r e 8.8, i n d i c a t i n g how t h e a n g u l a r c o r r e l a t i o n s W(Tr,t) and W ( 7 r / 2 , t ) a r e r o u t e d t o s e p a r a t e h a l v e s o f t h e m u l t i c h a n n e l a n a l y z e r memory.  The y-ray e n e r g i e s were s e l e c t e d i n t h e  slow s i d e o f t h e c i r c u i t by a t i m i n g s i n g l e c h a n n e l a n a l y z e r (TSCA). The t i m e - t o - a m p l i t u d e c o n v e r t e r (TAC) measured t h e time between t h e a r r i v a l o f t h e s t a r t  (173 keV) and s t o p (247 keV) p u l s e s ,  w h i l e t h e slow c o i n c i d e n c e l o g i c determined d e t e c t o r s t h e event o r i g i n a t e d .  interval  between which p a i r o f  The events were then r o u t e d t o s e p a r a t e  h a l v e s o f t h e m u l t i c h a n n e l a n a l y z e r memory.  The m u l t i c h a n n e l a n a l y z e r  would then p r o v i d e a d i r e c t d i s p l a y o f e m i s s i o n a n i s t r o p y a g a i n s t d e l a y time.  The prompt time r e s o l u t i o n o f t h i s system was 1.1 ns f o r ^ C o  gamma r a y s .  However, w i t h t h e energy windows s e t a p p r o p r i a t e l y f o r  the 173 keV and 247 keV gamma r a y s i n "'"^^Cd, t h i s d e t e r i o r a t e d t o 2.7 n s . By measuring W ( i T , t ) and W(ir/2,t) s i m u l t a n e o u s l y , no c o r r e c t i o n f o r t h e decay o f the source was n e c e s s a r y , and improved c o u n t i n g s t a t i s t i c s resulted.  The s c a l e r counts i n t h e slow  automatically provide the required normalizations. ensured  side-channels Gain  stabilizers  l o n g - t e r m s t a b i l i t y i n t h e l i n e a r p u l s e s from t h e a m p l i f i e r s and  a n t i - c o i n c i d e n c e requirements c o u n t i n g system.  were used t o e l i m i n a t e " c r o s s - t a l k " i n t h e  n  XL  0°  90' SLOW 1FAST  SLOWr  180° SLOW FAST  FAST  FASTI OR  — r  LA  LA  STARTI T.S.C.A Y2  1  SCALER  |SCALER \SLOW COIN  8.8.  ANTI  -4v  LIN. GATE  MIXER MCA 90°  MIXER MCA 180°  A s i m p l i f i e d block diagram o f the e l e c t r o n i c s  experiments.  perturbed  T. A . C  2  SLOW COIN  used i n t h e p r e s e n t t i m e - d i f f e r e n t i a l correlation  v  •HSCALER  LIN. GATE  1  Figure  T.S.C.A  T.S.C.A  tSTOP  angular  co  ----140The samples i n s o l u t i o n o r s o l i d form were c o n t a i n e d i n normal g l a s s n.m.r. tubes and kept a t 10-15°C by a stream o f c o l d a i r .  The  samples were a d j u s t e d t o g i v e a d i s i n t e g r a t i o n r a t e u s u a l l y o f t h e 4 o r d e r o f 4x10  4 - 6x10  counts per second i n o r d e r t o reduce t h e  chance c o i n c i d e n c e c o n t r i b u t i o n  to the true coincidence rate.  A t y p i c a l t i m e - d i f f e r e n t i a l perturbed angular c o r r e l a t i o n measurement t a k e s about 2 days.  The d a t a were c o r r e c t e d  e f f e c t s and f i n i t e t i m e r e s o l u t i o n .  The d i f f e r e n t i a l  f o r s o l i d angle  non-linearity  of t h e t i m e spectrum was measured w i t h random s t o p and s t a r t p u l s e s i n the TAC and found t o be e s s e n t i a l l y over 90% o f t h e time range. V i s c o s i t y measurements were performed on t h e p r o t e i n samples u s i n g a Ostwald v i s c o m e t e r .  The change i n s o l u t i o n v i s c o s i t y was  e f f e c t e d by a d d i n g p r e d e t e r m i n e d amount o f g l y c e r i n e .  The s o l i d  of p r o t e i n was o b t a i n e d by l y o p h i l i z a t i o n o f t h e p r o t e i n  form  solution.  -141CHAPTER IX  RESULTS AND  9.1  DISCUSSION  R E S U L T S - T i m e - D i f f e r e n t i a l P e r t u r b e d A n g u l a r C o r r e l a t i o n s Data  The r e c o n s t i t u t e d ^^InMPP-Mb was  s t u d i e d i n 0.05M sodium  b u f f e r , pH 7.0, and i n l y o p h i l i z e d powder form a t 12°C. of for  the aqueous sample was  changed  by adding g l y c e r i n e .  the t i m e - d i f f e r e n t i a l p e r t u r b e d a n g u l a r c o r r e l a t i o n  The  phosphate  viscosity  I n the  samples  (TDPAC) e x p e r i -  ments, no attempt was made t o ensure complete i n c o r p o r a t i o n o f ^"^InMPP I X i n t o apomyoglobin.  The excess apomyoglobin  s h o u l d pose no  difficulty  s i n c e o n l y t h o s e m y o g l o b i n m o l e c u l e s c o n t a i n i n g ^^"InMPP IX w i l l  con-  t r i b u t e t o the TDPAC s i g n a l . The TDPAC s p e c t r a f o r  111  I n M P P - M b i n aqueous b u f f e r w i t h and w i t h o u t  g l y c e r i n , and i n l y o p h i l i s e d powder form a r e shown i n F i g u r e s 9.1-9.3. R e s u l t s f o r the f r e e  1:L1  I n M P P I X i n d i m e t h y l formamide (DMF)  i n c h l o r o f o r m (CHCl^) a r e i l l u s t r a t e d are  least-square f i t s  r  where A  A  2  v  _  '  = -0.18.  C0R "  where Q  InTPP:Cl  The s o l i d  curves  2 2  (t)  w  e  r  e  determined from the measured  W(Tr/2,t)  (t\  22  1 : L 1  to the e x p e r i m e n t a l d a t a .  The p e r t u r b a t i o n f a c t o r s ^ W(iT,t) and  i n F i g u r e s 9.4-9.5.  and  _2  W(TT,t)-W(Tt/2,t)  A  W(TT,t)+2W(ff/2,t)  2  (6.5)  The d a t a were c o r r e c t e d f o r s o l i d a n g l e e f f e c t s w i t h  (9.1)  W V W  a r e the s o l i d a n g l e c o r r e c t i o n  factors.  For t h e aqueous samples, a t h e o r e t i c a l f i t t o t h e measured p e r t u r b a t i o n f a c t o r s was made u s i n g t h e f o l l o w i n g  G (t) 2 2  = (1-A) + Aexp(  t  function  /x )G (t) c  (9.2)  2  where G ( t ) i s t h e s t a t i c r e s u l t o f p o l y c r y s t a l l i n e powder. 2  a x i a l l y symmetric  F o r a non-  e l e c t r i c g r a d i e n t , r e p r e s e n t e d by t h e asymmetry, n  and a f i n i t e s p r e a d , <5 i n t h e quadrupole  i n t e r a c t i o n , G ( t ) i s g i v e n by 2  3  G„(t,n,6) = z  E b (n)exp(-%w T )exp(-J2co 6 t ) COS(OJ t ) (9.3) n n c n n n=o 2  2  2  2  2  2 The f a c t o r exv>(-h(ii T ) a l l o w s f o r f i n i t e time r e s o l u t i o n and t h e n c quadrupole f r e q u e n c i e s a r e g i v e n as  u (n) = f ( n ) u n n o  (9.4)  The f ( ) v a l u e s a r e o b t a i n e d from t h e d i a g o n a l i z a t i o n o f t h e n  non-symmetric f i e l d H a m i l t o n i a (141).  F o r n=o ( a x i a l symmetry) f ( l )  t a k e on t h e i n t e g r a l v a l u e s 1, 2, and 3. longer  For nfo, the ( ) w  n  n  a r  e no  harmonics. 2 The program MINUIT (153) was used t o m i n i m i z e t h e v a l u e o f x  t h e space o f f r e e parameters,  in  T , OJ , and 6 (the r e l a t i v e w i d t h C  q  parameter). For t h e s o l i d sample where t h e r e was no motion a t a l l (T -*»), t h e f o l l o w i n g f u n c t i o n was used t o f i t t h e TDPAC d a t a G ( t ) = G (t,n,6) 2 2  s  (9.5)  -143The f i t t e d v a l u e s o b t a i n e d  f o r T , r\, and 6 f o r each o f t h e case  a r e c o m p i l e d i n T a b l e 9.1  T a b l e 9.1.  Parameters d e r i v e d from a n a l y s i s o f TDPAC s p e c t r a f o r l l l l n M P P - M b s o l i d and i n aqueous b u f f e r s o l u t i o n .  A Lyophilized (solid)protein  1.0  Aqueous p r o t e i n c o n t a i n i n g 41% glycerine  0.98±0.01  Aqueous p r o t e i n without glycerine  0.96±0.01  T  (ns) c  00  «3  n  6  36±4  0.01±0. 01  0.37±0. 03  30±3  0.01±0. 01  0.36±0. 04  25±3  0.00±0. 01  0.42±0. 04  (MHz) o  0)  09 D  I  1  0.0  1  18.125  1  36.25  1  54.375  CHANNEL Figure 9 . 1 .  Time-differential  spectrum f o r ^ I n M P P - M b (pH = 7 . 0 )  with  percentage of 0.69ns  1  72.5  90.625  per channel.  1  126.88  NUMBER perturbed  i n aqueous  angular c o r r e l a t i o n  sodium phosphate  g l y c e r i n e added t o g i v e a f i n a l  41.  1  108.75  buffer  glycerine  1  145.0  1 0.0  1  18.125  1 54.375  1  36.25  1  CHANNEL Figure  9.2.  Time-differential  spectrum f o r  ^InMPP-Mb  (pH = 7 . 0 )  without  0.69ns  channel.  per  1  72.5  1 126.88  angular  correlation  108.75  NUMBER perturbed  i n aqueous  glycerine.  1  90.625  sodium phopshate  buffer  1  cn  0.0  1  18.125  36.25  1  54.375  CHANNEL Figure 9 . 3 .  Time-differential  spectrum for^^InMPP-Mb 0.69ns  per channel.  1  72.5  1  90.625  1  108.75  126.88  NUMBER perturbed  angular  i n the l y o p h i l i z e d  powder  correlation form.  145.0  F i g u r e 9.4.  Time-differential  spectrum f o r  ] 1 1  InMPP  IX  in  perturbed  angular  dimethylformamide.  correlation  lO  »8.i25  3B~25  54.375  72.5  90.625  108.75  126.88  CHANNEL NUMBER F i g u r e 9.5.  Time-differential  spectrum f o r ^ I n T P P  perturbed  in chloroform.  angular  correlation  145 D  -149-  9.2  DISCUSSION  9.2a  A n a l y s i s of the T l m e - D l f f e r e n t l a l Perturbed Angular C o r r e l a t i o n Spectra 111 The q u a l i t a t i v e appearance o f t h e l y o p h i l i z e d  ( F i g u r e 9.3) c o n f i r m s  InMPP-Mb TDPAC d a t a  t h a t "'"''"''"InMPP I X i s t i g h t l y bound t o m y o g l o b i n and  i s experiencing a s t a t i c quadrupolar  interaction.  The e x p e r i m e n t a l  does not show t h e normal c h a r a c t e r i s t i c p e r i o d i c b e h a v i o r G  22^^  ^  o r  s  t  a  t  i  c  curve  of t h e d i f f e r e n t i a l  i n t e r a c t i o n s as o c c u r r i n g i n p o l y c r y s t a l l i n e samples (see  F i g u r e 6.6 i n S e c t i o n 6.2b).  I n s t e a d t h e l y o p h i l i z e d p r o t e i n sample shows  f i r s t a r a p i d decay and then f l u c t u a t e s around t h e low v a l u e . p e r i o d i c observation i s probably  T h i s non-  due t o e f f e c t s a r i s i n g from t h e e x c i t e d  e l e c t r o n s h e l l a f t e r K - c a p t u r e d u r i n g t h e t r a n s i t i o n from "'""'""'"In t o t h e e x c i t e d "'""'"''"Cd s t a t e (see Appendix C) . s i m i l a r behavior  Lehmann and M i l l e r a l s o observed  f o r t h e two n o n - m e t a l l i c sources  In^O^  and In(OH)^, as  d i s c u s s e d i n S e c t i o n 6.2b. F i g u r e 9.2 g i v e s t h e TDPAC r e s u l t s f o r ''"''""'"InMPP-Mb i n aqueous buffer s o l u t i o n without  g l y c e r i n e a t 12°C.  The a n g u l a r  correlation  i s strongly perturbed.  T h i s i n d i c a t e s t h a t "'""'""'"InMPP IX i s bound and i s  i n a r e l a t i v e l y immobile environment ( i . e . , l o n g r o t a t i o n a l time).  correlation  As a c o n t r o l , TDPAC experiments were performed on f r e e "'""'""'"InMPP I X  ( i . e . , i n t h e absence o f p r o t e i n ) i n DMF and r e s u l t s a r e shown i n F i g u r e s 9.4 and 9.5. r e s u l t s obtained  1 1 : L  I n T P P : C l i n CHC1 . 3  The  I n c o n t r a s t t o t h e TDPAC  f o r t h e "'""'""''InMPP-Mb i n aqueous b u f f e r , t h e a n g u l a r  c o r r e l a t i o n i s e s s e n t i a l l y unperturbed or i s o t r o p i c f o r the f r e e '''''""''InMPP IX and "'""'"^InTPPrCl. molecular  T h i s o b s e r v a t i o n i m p l i e s much f a s t e r  r o t a t i o n f o r t h e l a t t e r case w h i c h l e a d s t o t h e much slower  e x p o n e n t i a l decay of a n i s o t r o p y .  -150The a t t e n t u a t i o n f a c t o r s samples  G 2 2  (t)  observed f o r the above s o l u t i o n  can be e x p l a i n e d i n terms of the i n f l u e n c e o f m o l e c u l a r r o t a t i o n  i n s o l u t i o n on a n i s o t r o p y . As d i s c u s s e d i n S e c t i o n 6.2a  i n Chapter VI,  Abragam and Pound showed t h a t f o r s m a l l molecules or i o n s o r i e n t e d i n d i l u t e s o l u t i o n s the d i f f e r e n t i a l  G 2 2  (t)  randomly  takes the form  of a simple e x p o n e n t i a l decay  G  2 2  (t)  =  exp(-A /t)  (6.6)  2  where  X  2  =  T00  (  e  2  q  Q  )  2  x  c  /  h  <->  2  6  I t i s c l e a r from Eq. 6.7 r o t a t i o n a l c o r r e l a t i o n time, x m o l e c u l e s ; T =10  "'""''sec.  that A c  2  7  i s d i r e c t l y p r o p o r t i o n a l to  which i s u s u a l l y s m a l l f o r s m a l l  Consequently, from Eqs. 6.6  and 6.7,  a short  x^ w i l l g i v e r i s e t o a s m a l l a t t e n t u a t i o n of the a n g u l a r c o r r e l a t i o n s in solution.  T h i s e x p l a i n s why  the a n i s o t r o p y of angular c o r r e l a t i o n s  remains l a r g e l y unperturbed f o r s m a l l molecules such as "'""''"'"InMPP IX and "'""'""'"InTPPrCl.  However, f o r l a r g e macromolecules  l i k e myoglobin  x^ as expected i s l a r g e , which accounts f o r the low G  2 2  2 2  ( t ) f o r the aqueous p r o t e i n sample without  i s s i m i l a r t o t h a t o b t a i n e d i n the l y o p h i l i z e d p r o t e i n sample. suggests a q u a s i s t a t i c i n t e r a c t i o n i n which G dependent.  2 2  (t) is itself  The t h e o r e t i c a l f i t to the data shows t h a t G  2 2  a c h a r a c t e r i s t i c minimum f o l l o w e d by a secondary maximum. i s smeared out, presumably  =  ( t ) v a l u e and  s t r o n g l y p e r t u r b e d a n g u l a r c o r r e l a t i o n f o r the aqueous p r o t e i n The form of G  (MW  by inhomogeneities i n the V ^  z  17,000), the  sample. glycerine This  time-  ( t ) f a l l s to The  latter  (component of  the e l e c t r i c f i e l d g r a d i e n t i n t h e z - d i r e c t i o n )  o r from t h e a f t e r -  e f f e c t s o f e l e c t r o n c a p t u r e , w h i c h wipe out t h e a n g u l a r A s l i g h t l y more s i g n i f i c a n t a t t e n t u a t i o n  correlations.  i s observed i n t h e s o l i d  case than i n aqueous sample.  However, t h e c l o s e r e l a t i o n s h i p between  the s o l i d and aqueous p r o t e i n  samples i s c o n s i s t e n t  w i t h the idea  t h a t ''"''"'''InMPP IX i s i n c o r p o r a t e d i n t o t h e a c t i v e - s i t e on t h e apomyoglobin. As d i s c u s s e d i n S e c t i o n 6.2a, t h e 3 - d i m e n s i o n a l m o l e c u l a r r o t a t i o n a l c o r r e l a t i o n time i s g i v e n by t h e e q u a t i o n below  4 R 3KT3  T  c=  5  ( 6  '  I t can be seen from Eq. 6.10 t h a t t h e d i r e c t r e l a t i o n s h i p between viscosity  1 0 )  and  ( ? ) s u g g e s t s t h a t c h a n g i n g t h e v i s c o s i t y o f t h e medium would  cause a c o r r e s p o n d i n g change i n a n i s o t r o p y o f t h e a n g u l a r I n t h e aqueous p r o t e i n  sample c o n t a i n i n g  41% g l y c e r i n e ,  correlations.  the increase  i n s o l u t i o n v i s c o s i t y l e a d s t o a l o n g e r r o t a t i o n a l c o r r e l a t i o n time for the protein.  T h i s s l o w e r m o l e c u l a r m o t i o n ( l o n g T ) causes a r a p i d  decrease i n the a n i s o t r o p y (Figure and  6.7.  9.1), i n a c c o r d a n c e w i t h Eqs. 6.6  The more pronounced G^it)  minimum s u g g e s t s a c l o s e r  between t h e l y o p h i l i z e d p r o t e i n and t h e aqueous p r o t e i n 41% 9.2b  glycerine  than t h e one w i t h no  The  sample  containing  glycerine.  Lease-Squares A n a l y s e s of t h e T i m e - D i f f e r e n t i a l Correlation  similarity  Perturbed Angular  Data  l e a s t - s q u a r e s a n a l y s e s of t h e TDPAC d a t a u s i n g v a r i o u s  f o r t h e t i m e - d i f f e r e n t i a l (^(O  include  (a) a pure s t a t i c  functions  interaction  which c o r r e s p o n d s t o Eq. 9.5, and (b) a s i n g l e s t a t i c i n t e r a c t i o n combined  w i t h r o t a t i o n a l d i f f u s i o n ( i . e . , m u l t i p l i c a t i v e combination of a s t a t i c and time-dependent i n t e r a c t i o n s ) as e x p r e s s e d by Eq. 9.2.  I t can be  seen from F i g u r e s 9.1-9.3 t h a t t h e t h e o r e t i c a l f i t s a r e adequate f o r t h e e a r l y TDPAC d a t a (<40 nsec) b u t a r e r a t h e r poor f o r l a t e r times  (>40 nsec)  T h e r e f o r e , f o r b o t h aqueous and s o l i d measurements, t h e e a r l y TDPAC d a t a s h o u l d be more r e l i a b l e and m e a n i n g f u l .  I n a d d i t i o n e a r l y TDPAC  d a t a has a g r e a t e r s t a t i s t i c a l a c c u r a c y and i n t r i n s i c a l l y more s i g n i f i c a n t i n f o r m a t i o n c o n c e r n i n g q u a d r u p o l a r whereas t h e l a t e r d a t a i s more prone t o e x p e r i m e n t a l The  contains  interactions, errors.  s o l i d c u r v e i n F i g u r e 9.3 r e p r e s e n t s t h e t h e o r e t i c a l f i t  c a l c u l a t e d u s i n g Eq. 9.5 f o r t h e l y o p h i l i z e d p r o t e i n TDPAC d a t a . The  curve i s c h a r a c t e r i s t i c of a s i n g l e s t a t i c quadrupolar  action.  inter-  Agreement w i t h t h e e a r l y e x p e r i m e n t a l d a t a i s good.  t h e r e q u i r e d v a l u e o f 6 ( d i s t r i b u t i o n i n quadrupole  frequency)  However, i n the  f i t was s u f f i c i e n t l y l a r g e t o wipe out a l l t h e f i n e s t r u c t u r e s a t l a t e r t i m e s as r e f l e c t e d by t h e poor agreement w i t h t h e l a t e r TDPAC data.  The f i t t e d asymmetry parameter, n=0.01±0.01 i s c o n s i s t e n t w i t h  the e x p e c t a t i o n t h a t t h e e l e c t r i c f i e l d g r a d i e n t around "^^"InMPP IX 111 s h o u l d be a x i a l l y symmetric i n t h e s q u a r e - p y r a m i d a l  IriMPP IX complex.  The TDPAC d a t a f o r b o t h t h e aqueous samples ( w i t h and w i t h o u t g l y c e r i n e ) were i n t e r p r e t e d on t h e b a s i s o f a c o m b i n a t i o n o f a s t a t i c and a time-dependent i n t e r a c t i o n mechanism.  The s o l i d c u r v e s i n  F i g u r e s 9.1 and 9.2 r e p r e s e n t t h e t h e o r e t i c a l c u r v e s c a l c u l a t e d u s i n g Eq. 9.2 f o r t h e two aqueous samples.  The good agreement w i t h t h e e a r l y  TDPAC d a t a , t o g e t h e r w i t h t h e f a c t t h a t t h e f i t t e d n=o, show t h a t t h e "^^InMPP IX i s indeed e x p e r i e n c i n g a s t a t i c i n t e r a c t i o n due t o i t s  -153-  s u r r o u n d i n g e l e c t r o n s , as w e l l as a time-dependent  interaction  a r i s i n g from t h e r o t a t i o n a l d i f f u s i o n o f t h e p r o t e i n m o l e c u l e . The  s i t u a t i o n s f o r b o t h t h e aqueous samples a r e s i m i l a r t o t h e  r e s u l t s d e r i v e d by M a r s h a l l and Meares (13) f o r slow m o l e c u l a r m o t i o n ( s e e S e c t i o n 6.2c). the f i t s a r e damped times  As i n t h e case o f t h e l y o p h i l i z e d  protein,  o u t t o g i v e an e s s e n t i a l l y f l a t l i n e s a t l a t e r  (>40 n s e c ) , a g a i n due t o t h e l a r g e r e q u i r e d v a l u e s o f <5. The  c u r v e s , however, a r e a b l e t o reproduce  t h e e a r l y TDPAC d a t a (<40 nsec)  accurately. The r e s u l t s o f t h e l e a s t - s q u a r e s a n a l y s e s c o m p i l e d  i n Table  9.1  show t h a t t h e v a l u e o f 0)^(3013 MHz) f o r t h e aqueous p r o t e i n c o n t a i n i n g 41% g l y c e r i n e i s comparable t o t h a t o f t h e l y o p h i l i z e d p r o t e i n (w =36±4 MHz). o  T h i s c l o s e r e l a t i o n s h i p between t h e s e two u> v a l u e s o  t o g e t h e r w i t h t h e f a c t t h a t t h e f i t t e d asymmetry parameter,n, i s z e r o , c o n f i r m s t h e c o r r e c t n e s s o f t h e TDPAC d a t a r e d u c t i o n . 9.2c  Comments on t h e R o t a t i o n a l C o r r e l a t i o n Times o f m y o g l o b i n determined  by t h e P e r t u r b e d A n g u l a r C o r r e l a t i o n Technique  As mentioned e a r l i e r , t h e 3 - d i m e n s i o n a l time  ( ) of a s p h e r i c a l molecule T  c  from t h e f o l l o w i n g  rotational  correlation  i n a l i q u i d medium can be o b t a i n e d  equation 3 r  4^R T  c  =  IRT"  5  , c i r \ \ ( 6  -  1 0 )  E q u a t i o n 6.10 can then be used i n two d i f f e r e n t ways t o compute T . The f i r s t method r e q u i r e s t h e v a l u e o f the Stokes r a d i u s ( R ) , g i v e n by t h e f o r m u l a below.  ( o r hydrodynamic)  -154—  =  R  (9  rot  where K = Boltzman c o n s t a n t , 1.38  ' > 6  x 10 " ^ e r g K ^  T = a b s o l u t e temperature n = asymmetry D  parameter  = r o t a t i o n a l d i f f u s i o n c o e f f i c i e n t , determined by  rot  J  sedimentation  experiments  The Stokes r a d i u s (R) f o r a number o f b i o l o g i c a l  macromolecules  (apomyoglobin, chymotrypsin, hemoglobins) were c a l c u l a t e d published D  rot  v a l u e s a c c o r d i n g to Eq. 9.6.  were then s u b s t i t u t e d The r e s u l t s a r e l i s t e d  i n t o Eq. 6.10 i n Table  using  These Stokes r a d i u s v a l u e s  to o b t a i n the c o r r e s p o n d i n g  values.  9.2.  The second method i n v o l v e s the assumption t h a t p r o t e i n m o l e c u l e s are r i g i d  unhydrated spheres, and each has a p a r t i a l s p e c i f i c volume 3  of 0.73  cm  /g.  E q u a t i o n 6.10  can then be r e w r i t t e n to g i v e Eq.  9.7  as shown below T  c  3 3~  =  KT  ( 6  = (volume/no.  in which  1 0 )  of m o l e c u l e s )  = (volume/gram)(gram/mole) (molecules/mole)  = vM N o  -  g KT  (9.7)  KT  M  =  molecular weight of protein  v  =  p a r t i a l s e p c i f i c volume, 0.73 cm~Vg  3,  -J55—  4  R Kl  N  o  £  3  =  volume o f a s p h e r i c a l  molecule  =  Avogadro's number, 6.023 x 10  =  v i s c o s i t y of w a t e r ; 0.01005 g cm ^ sec a t 20°C; 0.012362 g cm  sec  23  molecule/mole  a t 12°C;  (c.g.s.) 0.008937 g cm  a t 25°C. The v a l u e s of T determined  c  f o r t h e same b i o l o g i c a l macromolecules were  u s i n g Eq. 9.7  A comparison  and a r e a l s o shown i n T a b l e  of t h e s e two s e t s of  9.2.  v a l u e s i n T a b l e 9.2  shows  t h a t t h e 3 - d i m e n s i o n a l r o t a t i o n a l c o r r e l a t i o n times c a l c u l a t e d u s i n g Eq. 9.7  a r e a l l s m a l l e r than t h e c o r r e s p o n d i n g v a l u e s u s i n g Eq.  T h i s can be r e a d i l y e x p l a i n e d by r e v i e w i n g the assumptions the d e r i v a t i o n s of those two e q u a t i o n s .  I n t h e case of Eq.  p r o t e i n m o l e c u l e s a r e assumed t o be r i g i d unhydrated f i x e d p a r t i a l s p e c i f i c volume.  T h i s assumption  6.10.  made i n 9.7,  spheres w i t h a  i s really quite a  d e p a r t u r e from t h e a c t u a l s i t u a t i o n s i n c e p r o t e i n m o l e c u l e s a r e known t o be e x t e n s i v e l y h y d r a t e d . Eq. 6.10,  A much b e t t e r a p p r o x i m a t i o n i s made i n  w h i c h i s based on the e f f e c t i v e m o l e c u l a r s i z e of the p r o t e i n  from i t s hydrodynamic r a d i u s (R).  Hence the h i g h e r x^ v a l u e s computed  by the l a t t e r method s h o u l d be a more a c c u r a t e r e p r e s e n t a t i o n o f the proteins' molecular T a b l e 9.2  motions.  a l s o i n c l u d e s some l i t e r a t u r e T  values obtained  from  c ESR and f l u o r e s c e n c e d e p o l a r i z a t i o n (F.D) measurements.  These r e s u l t s  a r e s i g n i f i c a n t l y d i f f e r e n t from the c o r r e s p o n d i n g x^_ v a l u e s computed u s i n g t h e hydrodynamic r a d i u s w i t h the Debye e q u a t i o n 6.10.  The  Table 9.2  Protein  Myoglobin  R o t a t i o n a l c o r r e l a t i o n times, x  Molecular Weight 17,000  Temperature CO 25 20 12  Chymotrypsin  25,000  20  Hemoglobin (tertamer)  66,000  20 12  Alpha2 (Hb-dimer)  33,000  Beta (Hb-dimer)  33,000  2  a.  Adapted from J . Yguerabide P.A.C. v a l u e s .  b.  From Ref. 155.  c.  From Refs. 156 and 157.  20 12 20 12  and Stokes r a d i i (R), of some p r o t e i n s  D„„ ,, , 2" . (era /s) 2 0  11.30x10  R(A) from Eq.9.6  b 18.9  T (nsec)from Eq. 9.7 tt..5 5..1 6..5  T ( n s e c ) * T (nsec)from from Eq.6..10° ESR  -7 8.5x10  8.5x10  C  6.1 7.0 8.9  7..5  6.9x10  T  8.3  12±2  f  16.0  26±2  8  20.0  30.9  19..9 25.,2  30.8 38.9  25.1  9. 9 12..6  16.5 20.8  12.3  25.1  9. 9 12. 6  16.5 20.8  12.3  d  ( r e f . 154). Values were c o r r e c t e d to conform w i t h the usual n.m.r., ESR and the  d. e From Ref. 158. f  From Shimshick et a l . , ( r e f . 159).  g  From McCallery et a l . , ( r e f . 160)  *  Assumed R i s constant.  (nsec)from F.D.  3  -151-  disagreement observed may be due t o t h e added e f f e c t of i n t e r n a l r o t a t i o n a l m o t i o n of the s p i n - l a b e l o r f l u o r e s c e n t - l a b e l , i n a d d i t i o n to  the m o l e c u l a r t u m b l i n g m o t i o n of t h e whole l a b e l l e d p r o t e i n . In  common w i t h o t h e r l a b e l l i n g t e c h n i q u e s , t h e P.A.C. method  s u f f e r s from the problem of d i s t i n g u i s h i n g l o c a l i s e d m o t i o n of probe itself  from the m o t i o n of the whole m o l e c u l e .  P.A.C. d e t e r m i n a t i o n o f  However, i n t h e p r e s e n t  of m y o g l o b i n , t h i s o b s t a c l e was  by r e c o n s t i t u t i n g t h e p r o t e i n .  overcome  The f e r r o - p r o t o p o r p h y r i n IX (heme)  was d i r e c t l y r e p l a c e d w i t h i n d i u m - I l l m e s o p r o t o p o r p h y r i n IX. t h i s way,  In  the r a d i o i s o t o p e , i n d i u m - I l l was i n t r o d u c e d i n t o t h e  a c t i v e s i t e of the p r o t e i n .  The X-ray s t r u c t u r e of m y o g l o b i n (74,  95) shows t h a t the heme f i t s t i g h t l y i n t o the a c t i v e s i t e .  Since  ^ ^ I n M P P IX i s s t r u c t u r a l l y s i m i l a r t o f e r r o - p r o t o p o r p h y r i n IX, t h e r e s h o u l d be v e r y l i t t l e f r e e r o t a t i o n o f t h e ^ ^ I n M P P IX a t the a c t i v e s i t e of the r e c o n s t i t u t e d m y o g l o b i n .  F u r t h e r m o r e , the i n d i u m -  l a b e l l e d p r o t e i n i s v e r y l i k e l y to r e t a i n i t s n a t i v e conformation, i n c o n t r a s t t o o t h e r l a b e l l i n g t e c h n i q u e s i n w h i c h t h e r e i s always u n c e r t a i n t y as t o how much the l a b e l d i s t o r t s the s t r u c t u r e of the protein being studied.  C o n s e q u e n t l y , s i n c e no a d d i t i o n a l  flexibility  i s i n t r o d u c e d a t the l a b e l attachment s i t e i n the P.A.C. method, the present determination of  f o r m y o g l o b i n i s p r o b a b l y more a c c u r a t e  than f l u o r e s c e n c e d e p o l a r i z a t i o n o r ESR d e t e r m i n a t i o n s . T a b l e 9.3 g i v e s the r o t a t i o n a l c o r r e l a t i o n times (T ) o f m y o g l o b i n and  InMPP IX r e c o n s t i t u t e d m y o g l o b i n a t v a r i o u s tem-  p e r a t u r e s and v i s c o s i t i e s .  I t i s i m p o r t a n t t o p o i n t out t h a t the  r o t a t i o n a l c o r r e l a t i o n t i m e s c a l c u l a t e d from Eq. 6.19  a t 12°C  based on the Stokes r a d i u s (R) of the p r o t e i n a t 20°C.  These  were values  Rotational correlation times, T of myoglobin and InMPP reconstituted myoglobin at various temperatures and viscosities (References cited i n Table 9.2) c  Protein  Myoglobin  Temperature (°C)  25 20 12  Viscosity a)(gcm s'h  T (nsec)from* Eq. 6.10 c  l  0.0089 0.0100 0.0123  6.1 7.0 8.9  inMPP-Mb i n 0.05M phosphate buffer, pH7.0 without glycerine 12  0.013  9.3  InMPP-Mb i n 0.05M phosphate buffer, pH7.0 with glycerine to give a f i n a l glycerine percentage of 41% 12  0.049  T (nsec)from P.A.C. c  T (nsec)from F.D. C  8.3  co  m  16  (11.5)  +  111  35.2  22  O  *  Calculated with R = 18.9A (from Table 9.2) at 20°C, and this value is assumed to remain the same at the other temperatures and v i s c o s i t i e s . Calculated assuming linear relationship between x  c  and viscosities (from the Debye Equation, Eq. 6.10).  -159s h o u l d be l a r g e r than i n d i c a t e d i n T a b l e  9.3, s i n c e m y o g l o b i n i s  expected  a t 12°C and thus have a  t o be more e x t e n s i v e l y h y d r a t e d  l a r g e r e f f e c t i v e molecular  radius (R).  R e s u l t s f o r ^^InMPP-Mb i n aqueous b u f f e r w i t h o u t g l y c e r i n e show t h a t t h e v a l u e o f T (P.A.C.) i s g r e a t e r than t h a t c a l c u l a t e d c  from t h e Debye e q u a t i o n experimental  (Eq. 6.10) by a f a c t o r o f 1.7.  This  higher  i" (P.A.C.) can be e x p l a i n e d by a l l o w i n g f o r a l a r g e r c  e f f e c t i v e molecular  r a d i u s (R) t h a n was used i n t h e Debye d e t e r m i n a t i o n .  A l t e r n a t e l y , the d i f f e r e n c e s i n  may r e f l e c t t h e f a c t t h a t m y o g l o b i n  i s not a s p h e r i c a l m o l e c u l e as i s assumed i n t h e Debye c a l c u l a t i o n . The d e v i a t i o n from a p e r f e c t sphere causes an i n c r e a s e i n the s u r f a c e a r e a t o volume r a t i o f o r m y o g l o b i n , thus i n c r e a s i n g r o t a t i o n a l and d e c r e a s i n g t h e r a t e o f m o l e c u l a r  friction  tumbling or T .  I t i s c l e a r from T a b l e 9.3 t h a t i n o r d e r t o compare t h e experimental to convert  T (P.A.C.) v a l u e w i t h T ( f l u o r e s c e n c e ) , i t i s n e c e s s a r y c  C  the only a v a i l a b l e  ( f l u o r e s c e n c e ) v a l u e a t 25°C t o 12°C.  T h i s c o n v e r s i o n was done by assuming t h a t  v a r i e s l i n e a r l y over a  range o f v i s c o s i t i e s , as was demonstrated e x p e r i m e n t a l l y f o r oxyhemog l o b i n by M c C a l l e r y e t . a l . , ( 1 6 0 ) ( F i g u r e 9.6).  The  values i n  F i g u r e 9.6 a r e p l o t t e d a g a i n s t t h e known v i s c o s i t i e s f o r t h e s u c r o s e c o n c e n t r a t i o n s used.  S i n c e t h e v i s c o s i t y o f water i n c r e a s e s by a f a c t o r  of 1.38 on l o w e r i n g t h e temperature from 25°C t o 12°C, t h e  (fluorescence)  of 9.3 nsec a t 25°C s h o u l d be i n c r e a s e d by t h e same f a c t o r t o 11.5 nsec a t 12°C.  The T ( f l u o r e s c e n c e ) a t 12°C i s lower c  T ( P . A . C . ) v a l u e o f 16 n s e c . c  than t h e e x p e r i m e n t a l  T h i s disagreement may a g a i n be due t o t h e  i n t e r n a l r o t a t i o n o f the f l u o r e s c e n t - l a b e l , i n a d d i t i o n to the molecular m o t i o n o f t h e whole p r o t e i n .  The p r e s e n t  s t u d y does however, e f f e c t i v e l y  -160-  200 r  D poise . y( , )x10 J  K  Figure 9.6.  Rotational  oxyhemoglobin Form M c C a l l e r y  c o r r e l a t i o n time  as a f u n c t i o n et  a l . ,  (ref.  of  n/T 160).  in  i n nanoseconds  poise/°K.  for  -161demcmstrate  t h a t the P.A.C. t e c h n i q u e can be used t o y i e l d  r o t a t i o n a l c o r r e l a t i o n times f o r The  proteins.  Debye model f o r r e l a x a t i o n (Eq. 6.10)  r o t a t i o n a l c o r r e l a t i o n time s h o u l d be protein solution containing glycerine.  increased  p r e d i c t s that i n the  reconstituted  41% g l y c e r i n e r e l a t i v e to t h a t w i t h o u t  s h o u l d expect an  16 nsec a t 0% g l y c e r i n e  As  increase  (5 = 0.013  (£ = 0.049 g cm  remains c o n s t a n t .  g cm"  C  t o 60 nsec at  41%  * s * ) , assuming the Stokes r a d i u s  (R)  1  s" ) 1  i s e v i d e n t from T a b l e 9.3,  the  experimental  c  at high concentrations  and  i n the T (P.A.C.) v a l u e of  T (P.A.C.) v a l u e at 41% g l y c e r i n e case i s o n l y 22 nsec.  However,  of g l y c e r i n e the number of f r e e w a t e r m o l e c u l e s  a v a i l a b l e f o r s o l v a t i n g the p r o t e i n i s r e d u c e d . hydration  the  A c c o r d i n g t o the d i r e c t l i n e a r r e l a t i o n s h i p between  v i s c o s i t y , one  glycerine  reasonable  sphere i s s m a l l e r  I n o t h e r words,  r e s u l t i n g i n a smaller  the  e f f e c t i v e molecular  3 radius T  c  (R).  Since  i s proportional  t o R £, any  more s i g n i f i c a n t l y than an e q u i v a l e n t  change i n R w i l l a f f e c t  change i n £.  Hence, a d e c r e a s e  i n the h y d r a t e d r a d i u s of the r e c o n s t i t u t e d m y o g l o b i n ( i n aqueous b u f f e r c o n t a i n i n g 41% g l y c e r i n e ) would cause a more s u b s t a n t i a l r e d u c t i o n i n the T v a l u e than can be compensated f o r by the c o r r e s p o n d i n g c increase  i n the v i s c o s i t y f a c t o r  (£)•  U s i n g the Debye e q u a t i o n (Eq. 6.10), the d e c r e a s e i n the e f f e c t i v e radius  of ^^"InMPP-Mb i n 41% g l y c e r i n e s o l u t i o n can  d e t e r m i n e d from the e x p e r i m e n t a l T ( P . A . C . ) v a l u e s . c  s u g g e s t s t h a t , i n 41% radius  This c a l c u l a t i o n  g l y c e r i n e s o l u t i o n , the e f f e c t i v e m o l e c u l a r  f o r m y o g l o b i n i s 72% as b i g as i n the absence of  (Table 9.4).  be  I t s h o u l d be s t r e s s e d , however, t h a t Eq.  an o v e r s i m p l i f i c a t i o n , s t r i c t l y a p p l i c a b l e o n l y  glycerine 6.10  represents  to i s o t r o p i c m o t i o n of  -162spherical molecules. f l a t t e n e d sphere  As p o i n t e d b e f o r e , myoglobin  i s more o f a  (74,95), and t h i s f a c t was not t a k e n i n t o  account  i n the preceding c a l c u l a t i o n s .  T a b l e 9.4  Stokes r a d i u s o f  InMPP-Mb c a l c u l a t e d from t h e Debye  model o f r e l a x a t i o n u s i n g e x p e r i m e n t a l T (P.A.C.) v a l u e s  Protein  Temp. (°C)  T (P.A.C.)  Viscosity(5) (g c m s ) _ 1  c  Stokes  (nsec)  _ 1  Radius(R)  (A)  li:L  InMPP-Mb i n phosphate b u f f e r without g l y c e r i n e  12  0.013  16  22.6  InMPP-Mb i n phosphate b u f f e r c o n t a i n i n g 41% glycerine  12  0.049  22  16.2  i:L1  + Eq. 6.10 was r e a r r a n g e d to g i v e  R  :  +  3 |3T kT  "\hfr  I n c o n c l u s i o n , t h e p r e s e n t study e f f e c t i v e l y demonstrates  that the  P e r t u r b e d A n g u l a r C o r r e l a t i o n (P.A.C.) method i s an a t t r a c t i v e , v i a b l e technique f o r o b t a i n i n g r o t a t i o n a l  c o r r e l a t i o n times f o r macromolecules. -12  Furthermore,  t h e h i g h s e n s i t i v i t y (as low as 10  M) combined w i t h  a b i l i t y f o r in v i v o e x p e r i m e n t a t i o n makes t h e P.A.C. method a u s e f u l approach  f o r examining o t h e r systems o f p h y s i o l o g i c a l i m p o r t a n c e .  In  a d d i t i o n , information concerning molecular behavior of p r o t e i n s i n s o l i d and l i q u i d s t a t e s can be o b t a i n e d . has been extended  t o hemoglobin,  Finally, a similar  study  i n view o f t h e h i g h l e v e l o f i n t e r e s t  -163-  and c o n t i n u i n g debate on P e r u t z ' s s t e r e o c h e m i c a l mechanism f o r oxygenbinding cooperativity.  -164APPENDIX A CO-OPERATIVE EFFECT OF REVERSIBLE OXYGENATION IN HEMOGLOBIN  Hemoglobin (Hb) and m y o g l o b i n (Mb) Hemoglobin, w h i c h i s c o n t a i n e d c a r r i e r i n blood.  are oxygen c a r r i e r s i n v e r t e b r a t e s .  i n r e d b l o o d c e l l s , s e r v e s as the oxygen  On the o t h e r hand, myoglobin f a c i l i t a t e s the t r a n s p o r t  o f oxygen i n muscle and a l s o s e r v e s as a r e s e r v e s u p p l y of oxygen i n t h a t tissue. The  shape of the oxygen d i s s o c i a t i o n curve of hemoglobin i s s i g m o i d a l  whereas t h a t of myoglobin i s h y p e r b o l i c ( F i g u r e A . l ) .  T h i s curve i s  o b t a i n e d by p l o t t i n g the f r a c t i o n a l s a t u r a t i o n w i t h oxygen v e r s u s  the  p a r t i a l p r e s s u r e of oxygen gas. However, the c h e m i s t r y and s t r u c t u r e of myoglobin is  c l o s e l y r e l a t e d to that  Then as P e r u t z  of the i n d i v i d u a l s u b u n i t s o f hemoglobin.  (6) posed the question,'Why i s i t not good enough f o r the  r e d c e l l t o c o n t a i n a s i m p l e oxygen c a r r i e r such as m y o g l o b i n " . The  p a r t i a l pressure  F i g u r e s A . l and A.2,  i n the l u n g s i s ^100  b o t h Hb and Mb  mm  Hg, and as e v i d e n t  from  are almost s a t u r a t e d w i t h oxygen. In  the venous b l o o d , as i n the t i s s u e s , much o f the oxygen has been r e l e a s e d from Hb whereas the m y o g l o b i n i s s t i l l s a t u r a t e d w i t h oxygen. For example a t 20 mm  Hg,  the s a t u r a t i o n of hemoglobin i s about 40% w h i l e i n m y o g l o b i n  i t i s about 80%  (see F i g u r e A . l ) .  The  sigmoid  g r e a t e r f r a c t i o n o f oxygen i s r e l e a s e d one.  Although  shape o f the curve means a  more r e a d i l y than the h y p e r b o l i c  Hb and Mb both b i n d oxygen, but Hb r e l e a s e s i t more r e a d i l y  i n o r d e r t h a t the t i s s u e s be a d e q u a t e l y  s u p p l i e d w i t h oxygen.  r e l e a s e s oxygen at a much lower p a r t i a l p r e s s u r e , and o x y g e n - c a r r i e r , the t i s s u e s would be  Myoglobin  i f Mb were the  asphyxiated.  Hemoglobin can be d i s s o c i a t e d i n t o i t s c o n s t i t u e n t s u b u n i t s ,  and  -]65-  experiments  have demonstrated t h a t t h e i n d i v i d u a l s u b u n i t s o f Hb do not  behave d i f f e r e n t l y from myoglobin. oxygen-binding  The d i f f e r e n c e between Hb and Mb i n  p r o p e r t y must then be a s s o c i a t e d w i t h t h e i n t e r a c t i o n s  between t h e s u b u n i t s , w h i c h somehow a l t e r t h e shape o f t h e e q u i l i b r i u m curve.  T h i s i n t e r a c t i o n i s o f t e n known as t h e "heme-heme  interaction"  (126).  This expression simply states that there are f u n c t i o n a l  interactions  between heme groups and does not i m p l y d i r e c t i n t e r a c t i o n s o r even t h a t t h e i n t e r a c t i o n s occur between t h e heme groups i n t h e same m o l e c u l e . The s i g m o i d shape o f t h e o x y g e n - d i s s o c i a t i o n c u r v e o f hemoglobin i m p l i e s t h a t the a f f i n i t y o f oxygen i n c r e a s e s w i t h the i n c r e a s e i n oxygen s a t u r a t i o n w h i c h c l e a r l y demonstrates t h e e x i s t e n c e o f a c o - o p e r a t i v e e f f e c t among t h e f o u r s u b u n i t s .  I n o t h e r words, the b i n d i n g o f oxygen  to t h e heme group o f one s u b u n i t has t h e e f f e c t o f i n c r e a s i n g t h e oxygen a f f i n i t y o f t h e n e i g h b o u r i n g s u b u n i t s o f t h e same m o l e c u l e . P e r u t z (6,28,29) proposed a s t e r e o c h e m i c a l i n t e r p r e t a t i o n o f t h e c o - o p e r a t i v e e f f e c t s i n hemoglobin. o r i g i n a l papers s h o u l d be c o n s u l t e d .  For t h e mechanism of t h i s  effect,  -166-  Figure A . l . B hemoglobin.  Oxygen e q u i l i b r i u m c u r v e s o f A myoglobin and  -167.-  APPENDIX B  TETRAPHENYLPORPHINE DIACID  T e t r a p h e n y l p o r p h i n e i s v i o l e t i n c h l o r o f o r m b u t green i n g l a c i a l acetic acid.  The former c o l o r i s a t t r i b u t e d t o t h e p o r p h y r i n f r e e base  ( F i g u r e B . l ) and t h e l a t t e r t o t h e f o r m a t i o n o f d i a c i d s p e c i e s ( F i g u r e B.2) . The p o r p h i n a t o core o f t e t r a p h e n y l p o r p h i n e f r e e base e x h i b i t s a marked d e v i a t i o n  from p l a n a r i t y o f t h e p o r p h i n a t o c o r e  (32,43).  S t r u c t u r a l s t u d i e s (32,43) have shown t h e p h e n y l groups a r e almost p e r p e n d i c u l a r t o t h e mean p l a n e o f t h e p o r p h y r i n r i n g because o f i n t e r a c t i o n s between t h e p h e n y l hydrogens and t h e o u t e r p y r r o l e  hydrogens.  T h i s arrangement o f t h e p h e n y l groups p r e v e n t s any Tr i n t e r a c t i o n between the benzene TT system and t h e h i g h l y c o n j u g a t e d p o r p h y r i n system; sample remains  thus t h e  violet.  I n t h e d i a c i d o f t e t r a p h e n y l p o r p h i n e , t h e p o r p h i n a t o c o r e i s even more h i g h l y d i s t o r t e d due t o v a n d e r Waals and Columbic the f o u r i n n e r hydrogen atoms.  r e p u l s i o n s of  In c o n t r a s t to the porphyrin free  the t i l t e d p o r p h i n e s k e l e t o n a l l o w s t h e phenyl groups t o r o t a t e  base,  toward  the mean p o r p h y r i n p l a n e making a d i h e d r a l a n g l e o f 21° w i t h i t ( F i g u r e B.3).  I n t h i s c a s e , s t r o n g ir i n t e r a c t i o n between the p o r p h y r i n and  p h e n y l IT e l e c t r o n s g i v e s r i s e t o t h e green c o l o r o f t h e d i a c i d s p e c i e s .  -168-  Figure B.3.  Geometries f o r porphyrin d i a c i d s .  -169APPENDIX C THE  EFFECT OF EXCITATION OF THE ELECTRON SHELL ON ANGULAR CORRELATION  D u r i n g r a d i o a c t i v e decay such as through a y-y  cascade,  there  i s a p o s s i b i l i t y t h a t a " h o l e " i s c r e a t e d i n one of the i n n e r e l e c t r o n (K) s h e l l s , w i t h a r e s u l t i n g atomic  s h e l l i n an e x c i t e d s t a t e .  After  the f o r m a t i o n o f the K - h o l e , the atomic s h e l l and n u c l e u s tend to r e t u r n t o t h e i r ground s t a t e s through y-y Auger e l e c t r o n s and X - r a y s . can be charged.  cascade and  e m i s s i o n of  F o l l o w i n g the Auger p r o c e s s , the atom  I t has been shown e x p e r i m e n t a l l y (161-164) t h a t Cd  can a c q u i r e an average charge 7e.  The  electric field  o r i g i n a t i n g from  t h e s e e x c i t e d atomic s h e l l s w i l l p e r t u r b the a n g u l a r c o r r e l a t i o n .  The  p e r t u r b a t i o n depends on the mean l i f e of t h e e x i c t e d s t a t e ( x ^ ) and on t h e t r a n s i t i o n p r o b a b i l i t i e s f o r X-ray and Auger e m i s s i o n i n v a r i o u s -12 shells.  T h i s l i f e t i m e i s known to be v e r y s h o r t (about 10  metals.  I n the case of i n s u l a t o r s i t may  sec) f o r  be as l o n g as seconds.  i n t e r a c t i o n s t r e n g t h i s of the o r d e r o f 500 MHz  The  which means t h a t t h e r e  -9  w i l l be an i n f l u e n c e i f T * 1 0  sec or l o n g e r .  The T„ f o r the 247-KeV  N  e x c i t e d s t a t e o f Cd^^"  i s 1.25  c o r r e l a t i o n i s expected. been  N  x 10 ^ s e c , and a p e r t u r b a t i o n on  angular  No d e t a i l e d a n a l y s i s of t h i s e f f e c t has  yet  developed. A f t e r t h e e l e c t r o n c a p t u r e , the K-hole c r e a t e d can  r a p i d l y towards the outermost s h e l l and  i n the p r o c e s s g e n e r a t e s more  h o l e s w h i c h a l s o move outwards d u r i n g such m i g r a t i o n . i s t h e consequence o f Auger e f f e c t .  progress  T h i s movement  Once these h o l e s have reached  outermost s h e l l , they can decay o n l y s l o w l y .  the  I f t h e i r mean l i f e t i m e  -170-  i s l o n g enough, a v e r y s t r o n g p e r t u r b a t i o n on t h e a n g u l a r c o r r e l a t i o n o f t e n r e s u l t e d . I f the  p e r t u r b a t i o n i s o f s u f f i c i e n t magnitude, t h e  c o r r e l a t i o n can be a t t e n u a t e d below t h e hard c o r e v a l u e (minimum v a l u e ) . The a t t e n t u a t i o n depends on t h e s u r r o u n d i n g atomic environment  which  can be an i n s u l a t o r o r a c o n d u c t o r . I n t h e case i n which t h e r a d i o a c t i v e atom i s i n c o r p o r a t e d i n a substance o f h i g h c o n d u c t i v i t y ( i . e . t h e presence o f c o n d u c t i o n band), the e l e c t r o n s h e l l e x c i t a t i o n f o l l o w i n g t h e K-^capture remains f o r a -12 short period  ( l e s s than 10  s e c ) . The n u c l e u s i s thus remained un-  p e r t u r b e d by t h e e l e c t r i c f i e l d s a r i s i n g from such e x c i t e d  shells.  The presence o f t h e c o n d u c t i o n band a l l o w s r a p i d f r e e f l o w o f e l e c t r o n s to " f i l l " state.  t h e h o l e s , and thus l e a v e s t h e r a d i o a c t i v e atom i n i t s ground  T h i s h o l d s t r u e whether t h e c o n d u c t o r c a r r y i n g t h e r a d i o n u c l e u s  i s i n a s o l i d o r l i q u i d medium. I n c o n t r a s t , i f the r a d i o a c t i v e atom i s i n c o r p o r a t e d i n a s o l i d i n s u l a t o r , t h e absence o f a c o n d u c t i o n band i n t h e r a d i o i s o t o p e ' s environment  l e a d s t o a l o n g decay time o f t h e h o l e s i n t h e outermost —8  shell  (>>10  s e c ) . C o n s e q u e n t l y , t h e i n s u l a t o r s o u r c e s h o u l d be  expected t o be a t t e n t u a t e d . However, t h e s i t u a t i o n i s more complex when t h e i n s u l a t o r c a r r i e r i s i n s o l u t i o n .  The i n f l u e n c e o f t h e K-  c a p t u r e on t h e a n g u l a r c o r r e l a t i o n s now depends on t h e r o t a t i o n a l c o r r e l a t i o n time (x  ) and t h e i n t e r a c t i o n s between t h e i n s u l a t o r c  molecules i n s o l u t i o n .  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